Isolated DNA encoding cullin regulators ROC1 and ROC2, isolated proteins encoded by the same, and methods utilizing the same

ABSTRACT

The present invention provides isolated polynucleotide sequences encoding the proteins ROC1 and ROC2, the isolated proteins themselves, expression vectors containing at least a fragment of the ROC1 and ROC2 polynucleotide sequences, and host cells comprising the same. Methods of producing the ROC1 and ROC2 proteins are also disclosed, and methods of detecting the polynucleotides in samples are included in this invention, as are antibodies to the ROC1 and ROC2 proteins and antisense molecules complementary to polynucleotides encoding the same. The present invention further includes methods for screening bioactive agents that are capable of binding to a ROC protein, methods of screening bioactive agents capable of interfering with the binding of ROC proteins, and methods of screening bioactive agents capable of modulating the activity of a ROC protein. Such screening methods are capable of identifying compounds that have pharmacological. Pharmaceutical formulations comprising such pharmacologically active compounds and methods of administering the same are an additional aspect of this invention.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/127,261, filed Mar. 31, 1999, and U.S. Provisional Application No.60/166,927, filed Nov. 22, 1999. Both Provisional Applications areincorporated herewith by reference in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under grant number RO1CA65572-01 from the National Institutes of Health. The United Statesgovernment has certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences ofcullin regulators that are associated with ubiquitin ligase activity,and to methods utilizing these sequences.

BACKGROUND OF THE INVENTION

The ubiquitin-dependent proteolytic process regulates many short livedintracellular proteins, whose concentrations change promptly as theresult of alterations in cellular physiological conditions. SeeHochstrasser, M. et al. (1996) Annu. Rev. Genet. 30, 405–439; King, R.W., et al. (1996) Science 274, 1652–1659; Hershko, A. et al. (1997)Curr. Opin. Cell Biol. 9, 788–799. In addition to performing“housekeeping” functions such as homeostasis and the removal ofmisfolded proteins, this proteolytic process is involved in thedegradation of many regulatory proteins, such as cyclins, CDKinhibitors, transcription factors, and signal transducers. In brief,ubiquitin-mediated proteolysis begins with activation of ubiquitin, a76-amino acid protein expressed in all eukaryotic cells, in anATP-dependent manner by an ubiquitin-activating enzyme (E1 or Uba). Theactivated ubiquitin forms a high energy thiolester bond with E1 and ispassed to a cysteine residue also via a thiolester bond within anubiquitin-conjugating enzyme designated as an E2 or Ubc. E2-linkedubiquitin is then transferred to a side chain amino group of a lysineresidue in the substrate to form a terminal isopeptide bond, eitherdirectly or often indirectly targeted by a ubiquitin ligase known as E3.Substrate proteins can be linked to a single ubiquitin(monoubiquitinated) or multiple ubiquitin molecules (polyubiquitinated).The significance of monoubiquitinated conjugates is not clear since theydo not appear to be short-lived. Successive covalent ligations ofadditional ubiquitins to the Lys 46 of the preceding ubiquitin via anisopeptide bond results in polyubiquitinated conjugates which arerapidly detected and degraded by the 26S proteosome. E3 is functionally,rather than structurally, defined as an ubiquitin ligase activity thatis both necessary and sufficient for transfer of ubiquitin from aubiquitin-charged E2 to a substrate, and is further believed to beinvolved in many polyubiquitination reactions by providing substratespecificity. Because most polyubiquitinated proteins areindiscriminately delivered to the 26S proteosome for degradation,elucidating the mechanism and regulation of E3 ligase activities hasbecome a critical issue central to the understanding of regulatedproteolysis.

The cullin family of proteins potentially form a large number ofdistinct E3s as indicated by the existence of a multi-gene family and bythe assembly of yeast CDC53 into at least three distinct E3 complexes:with SKP1-CDC4, with SKP1-GRR1 and likely with SKP1-MET30 to mediate theubiquitination of SIC1, CLN and SWE1 proteins, respectively. See, e.g.,Skowyra, D., et al., (1997) Cell 91, 209–219; Feldman, R.M.R. (1997)Cell 91, 221–230; and Kaiser, P. et al., (1998) Genes & Dev. 12,2587–2597. Through targeting different substrates, different cullinsfunction in a variety of diverse cellular processes. For example, CDC53is required for S phase entry (Mathias, N. et al., (1996) Mol. CellBiol. 16, 6634–6643; for coupling glucose sensing to gene expression andthe cell cycle (Li, F. N. and Johnston, M. (1997) EMBO J. 16,5629–5638;and possibly for activating mitotic CLB-CDC28 activity (Kaiser, P. etal., (1998) Genes & Dev. 12, 2587–2597). As set forth in more detailbelow, the C. elegans cul-I mutant displays a hyperplasia phenotype.Human CUL2 is associated with the tumor suppressor VHL (vonHippel-Lindau) implicated in the regulation of the stability ofhypoxia-induced mRNA (see Pause, A., et al. (1997) Proc. Natl. Acad. SciUSA. 94, 2156–2161; Lonergan, K. M. et al., (1998) Mol. Cell Biol. 18,732–741. Human CUL4A is implicated in oncogenesis by its genomicamplification and overexpression in breast cancers (Chen, L-C., et al.,(1998) Cancer Res. 58, 3677–3683), and deficiency of the cullin-relatedAPC2 results in mitotic arrest (Zachariae, W. et al., (1998) Science279, 1216–1219; Yu, H., et al., Current Biology 6, 455–466).

The knowledge of E3 ubiquitin ligases is presently limited. Among thefew characterized E3 ligases are the N-end rule ubiquitin ligaseE3α/Ubr1 that recognize proteins by binding to the basic or hydrophobicresidues at the amino-termini of substrate proteins (reviewed inVarshavsky, A. (1996) Proc. Natl. Acad. Sci U.S.A. 93, 12142–12149); theHECT (homologous to E6-AP carboxy terminus) domain proteins representedby the mammalian E6AP-E6 complex which functions as a ubiquitin-ligasefor p53 (see Scheffner, M. et al., (1993) Cell 75, 495–505; Huibregtse,J. M., et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2563–2567;Scheffner, M. et al. (1995) Nature 373, 81–83); and the APC(anaphase-promoting complex or cyclosome), a 20S complex that consistsof 8 to 12 subunits and is required for both entry into anaphase as wellas exit from mitosis (see King, R. W., Deshaies, Science 274,1652–1659).

The APC plays a crucial role in regulating the passage of cells throughanaphase by promoting ubiquitin-dependent proteolysis of many proteins.The APC destroys the mitotic B-type cyclin for inactivation of CDC2kinase activity and initiating cytokinesis. The APC is also required fordegradation of other proteins for sister chromatid separation andspindle disassembly, including the anaphase inhibitors PDS1 (Cohen-Fix,O., et al. (1996) Genes & Dev. 10, 3081–3093) and CUT2 (Funabiki, H., etal. (1996) Nature 381, 438–441), ASE1 (Juang, Y-L. et al. (1997) Science275, 1311–1314) and the cohesion protein SCC1P (Michaelis, C. et al.,(1997) Cell 91, 35–45). All known proteins degraded by the APC contain aconserved nine amino acid stretch commonly known as the destruction boxthat is necessary for their ubiquitination and subsequent degradation(Glotzer, M., et al. (1991) Nature 349, 132–138). Proteins that aredegraded during G1, ranging from G1 cyclins and CDK inhibitors totranscription factors, do not contain the conserved destruction box orany other common structural motif. Instead, substrate phosphorylationappears to play an important role in targeting their interaction with E3for subsequent ubiquitination. Genetic and biochemical analysis hasidentified in yeast an E3-like activity, dubbed as the SCF, that plays akey role in regulating G1 progression. The SCF consists of at leastthree subunits, SKP1, CDC53/cullin and an F-box containing protein, inwhich SKP1 functions as an adaptor to connect CDC53 to the F-box proteinwhich binds directly to the substrate (Feldman, R. M. R., et al., (1997)Cell 91, 221–230; Bai, C., et al. (1996) Cell 86, 263–274; Willems, A.R., (1996) Cell 86, 453–463; Verma, R. (1997) Science 278, 455–460;Skowyra, D., (1997) Cell 91, 209–219).

In a screen for mutants with excess postembryonic cell divisions in C.elegans, the gene cullin-1 (CUL1), was identified. Loss of function ofthis gene caused hyperplasia of all tissues as a result of the failureto properly exit from the cell cycle. See Kipreos, E. T., et al., (1996)Cell 85, 829–839. CULL represents an evolutionarily conserved multigenefamily that includes at least seven members in C. elegans, six inhumans, and three in budding yeast including Cdc53p (Kipreos, et al.,supra, and Mathias, N. et al., (1996) Mol. Cell Biol. 16, 6634–6643).Like yeast CDC53, human cullin 1 directly binds to SKP1 to form amulti-subunit complex with SKP2 (an F box protein), cyclin A and CDK2(Lisztwan, J. et al., (1998) EMBO J. 17, 368–383; Michel, J. and Xiong,Y. (1998) Cell Growth. Differ. 9, 439–445; Lyapina, S. A., et al. (1998)Proc. Natl. Acad. Sci. USA 95, 7451–7456; and Yu, Z. K. et al. (1998)Proc. Natl. Acad. Sci U.S.A. 95, 11324–11329), and can assemble intofunctional, chimeric ubiquitin ligase complexes with yeast SCFcomponents. Recently, a subunit of the mitotic APC E3 complex, APC2, wasfound to contain limited sequence similarity to CDC53/cullins(Zachariae, W. et al., (1998) Science 279, 1216–1219; Yu, H. et al.,(1998) Science 279, 1219–1222). These findings, together with the factthat no obvious structural similarity between other components of theSCF and APC complexes exists, underscore an important and conserved rolefor cullin proteins in ubiquitin-mediated proteolysis, possibly as anintrinsic partner of ubiquitin ligases. However, despite extensiveinvestigations of the APC and SCF E3 ligases, the nature of ubiquitinligases has thus far been elusive. It still remains to be determinedwhether there is a “ligase” in the APC and SCF. Whether the cullinproteins act as ubiquitin ligases to catalyze isopeptide bond formationor as scaffold proteins to bring together E2-Ub and substrates togetheris heretofore not described.

Equally important as the mechanism that determines the substratespecificity is the regulation of E3 ligases, which is presently poorlyunderstood. The activity of the APC is cell-cycle regulated, and activefrom anaphase until late G1. See Amon, A. (1994) Cell 77, 1037–1050;King, R., et al., (1995) supra; Brandeis, M. and Hunt, T. (1996) EMBO J.15, 5280–5289. The principle regulation is probably provided by subunitrearrangements such as CDC20 and CDH1 binding (Visintin, et al., (1997)Science 278, 460–463; Schwab, M. (1997) Cell 90, 683–693; Sigrist, S. J.and Lehner, C. F. (1997) Cell 90, 671–681; and Fang, G. (1998) Mol. Cell2, 163–171). Phosphorylation of certain subunits may also play animportant, but supplementary role (Lahav-Baratz, S., Proc. Natl. Acad.Sci. USA 92, 9303–9307; Peters, J.-M. et al. (1996) Science 274,1199–1201). Regulation of CDC53 and cullin-mediated E3 ligase activityduring interphase is heretofore not described.

SUMMARY OF THE INVENTION

The present inventors have identified a family of two closely relatedRING finger proteins, ROC1 and ROC2, that are similar to APC11, asubunit of the APC complex. ROC1 and ROC2 commonly interact with allcullin proteins, while APC11 specifically interacts with APC2. ROC1functions in vivo as an essential regulator of CDK inhibitor Sic1degradation by the SCF pathway. Additionally, the inventors have foundthat ROC-cullin constitutes the catalytic ubiquitin ligase. Although theinventors do not wish to be bound to any theory of the invention, it isthought that dimeric complexes of ROC1-cullins and APC11-APC2 functionas ubiquitin ligases during interphase and mitosis, respectively.

Accordingly, the invention provides an isolated polynucleotide sequenceencoding the protein ROC1. The polynucleotide sequence may be selectedfrom the group consisting of:

-   -   (a) DNA having the nucleotide sequence given herein as SEQ ID        NO:1 (which encodes the protein having the amino acid sequence        given herein as SEQ ID NO:2);    -   (b) polynucleotides that hybridize to DNA of (a) above (e.g.,        under stringent conditions) and which encode the protein ROC1;        and    -   (c) polynucleotides that differ from the DNA of (a) or (b) above        due to the degeneracy of the genetic code, and which encode the        protein ROC 1 encoded by a DNA of (a) or (b) above.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention further provides a protein or fragment thereof encoded bya polynucleotide as given above (e.g., the protein provided herein asSEQ ID NO: 2). Such proteins may be isolated and/or purified inaccordance with known techniques.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding ROC1 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides an antibody (e.g., a polyclonal antibody, amonoclonal antibody) which specifically binds to a protein as givenabove.

The invention provides an antisense oligonucleotide complementary to apolynucleotide sequence as given above and having a length sufficient tohybridize thereto under physiological conditions. DNA encoding such anantisense oligonucleotide, and a nucleic acid construct having apromoter and a heterologous nucleic acid operably linked to saidpromoter (wherein the heterologous nucleic acid is a DNA encoding suchan antisense oligonucleotide) is also an aspect of the invention.

The invention also provides a method for detecting a polynucleotidewhich encodes ROC1 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:1 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding ROC1 in the biological sample. Inone aspect, the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

Further, the invention provides an isolated polynucleotide sequenceencoding the protein ROC2. The polynucleotide sequence may be selectedfrom the group consisting of:

-   -   (a) DNA having the nucleotide sequence given herein as SEQ ID        NO:3 (which encodes the protein having the amino acid sequence        given herein as SEQ ID NO:4);    -   (d) polynucleotides that hybridize to DNA of (a) above (e.g.,        under stringent conditions) and which encode the protein ROC1        and    -   (e) polynucleotides that differ from the DNA of (a) or (b) above        due to the degeneracy of the genetic code, and which encodes the        protein ROC 1 encoded by a DNA of (a) or (b) above.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention further provides a protein or fragment thereof encoded bya polynucleotide as given above (e.g., the protein provided herein asSEQ ID NO: 4). Such proteins may be isolated and/or purified inaccordance with known techniques.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:4, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding ROC2 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides an antibody (e.g., a polyclonal antibody, amonoclonal antibody) which specifically binds to a protein as givenabove.

The invention provides an antisense oligonucleotide complementary to apolynucleotide as given above and having a length sufficient tohybridize thereto under physiological conditions. DNA encoding such anantisense oligonucleotide, and a nucleic acid construct having apromoter and a heterologous nucleic acid operably linked to saidpromoter (wherein the heterologous nucleic acid is a DNA encoding suchan antisense oligonucleotide) is also an aspect of the invention.

The invention also provides a method for detecting a polynucleotidewhich encodes ROC2 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:3 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding ROC2 in the biological sample. Inone aspect, the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

The invention provides methods for screening bioactive agents (the term“agent” and grammatical equivalents thereof being used interchangeablywith the term “compound” and the grammatical equivalents thereof) thatare capable of binding to a ROC protein, wherein a ROC protein and acandidate bioactive agent are combined. The binding of the candidatebioactive agent is then determined. Methods of screening bioactiveagents capable of interfering with the binding of ROC proteins, or ofmodulating the activity of a ROC protein, are also aspects of thepresent invention. Such screening methods are capable of identifyingcompounds that have pharmacological (pharmaceutical) activity.Pharmaceutical formulations comprising such pharmacologically activecompounds and methods of administering the same are another aspect ofthis invention. Yet another aspect of the present invention is the useof a pharmacologically active compound identified by the methodsdescribed herein for the manufacture of a medicament for theprophylactic or therapeutic use in a subject or host.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate that ROC1 interacts with members of thecullin family. In the experiments illustrated in FIG. 1A, yeast HF7ccells were co-transformed with plasmids expressing indicated proteins(key) and plated onto media lacking leucine and tryptophan (−LW) toverify the presence of both bait (Leu+) and prey (Trp+) plasmids; oronto media lacking leucine, tryptophan and histidine (−LWH) to assay forinteractions between bait and prey proteins.

FIG. 1B illustrates that ROC1 interacts with the C-terminal portion ofCUL1. HF7c yeast cells were co-transformed with plasmids expressingindicated proteins. Protein-protein interaction was assayed as describedin herein.

FIG. 2A sets forth the nucleotide sequence (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of human ROC1. The stop codon is indicatedby an asterisk.

FIG. 2B sets forth the nucleotide sequence (SEQ ID NO: 3) and the aminoacid sequence (SEQ ID NO: 4) of human ROC2. The stop codon is indicatedby an asterisk.

FIG. 2C illustrates the sequence comparison of ROC/APC11 family ofproteins from representative organisms: human (Hs, Homo sapiens), fruitfly (Dm: Drosophila melanogaster), nematodes (Ce: Caenorhabditiselegans), mouse ear cress (At: Arabidopsis thaliana), fission yeast (Sp:Schizosaccharomyces pombe), and budding yeast (Sc: Saccharomycescerevisiae). Residues that are identical among all sequences arepresented in bold type. The number in the bracket of certain sequencesindicates the length of the insertion omitted. The number preceding andfollowing each sequence indicates the position of the first amino acidresidue in each gene and the total length of each protein, respectively.

FIGS. 3A, 3B and 3C illustrate the in vivo association of ROC1 withcullins. In the experiments shown in FIG. 3A, [³⁵S]-methionine labeledlysates were prepared from HeLa cells transfected with plasmidsexpressing the indicated proteins. Lysates were divided into two equalamounts and immunoprecipitated with indicated antibodies and resolved bySDS-PAGE. For the experiments illustrated in FIG. 3B [³⁵S]-methioninelabeled, in vitro translated ROC1 (lane 1), mixture of ROC1 and CUL1(lane 2), or cell lysates from HeLa and Saos-2 cells wereimmunoprecipitated with anti-ROC1 antibody with (+) or without (−)pre-incubation of the competing ROC1 antigen peptide as indicated at thetop of each lane. After several washings, precipitates were resolved bySDS-PAGE.

For the experiments illustrated in FIG. 3C, total cell lysates preparedfrom HeLa cells were immunoprecipitated with indicated antibodies with(+) or without (−) competing antigen peptide. After SDS-PAGE, proteinswere transferred to nitrocellulose, and analyzed by Western analysisusing antibodies to CUL1 (lanes 1 to 4, top panel), to CUL2 (lanes 5 to8, top panel) or to ROC1 (bottom panel).

FIGS. 4A–4E illustrate that ROC2 and APC11 selectively interact withcullins and APC2. In the experiments shown in FIGS. 4A and 4B HF7c yeastcells were co-transformed with plasmids expressing human ROC2 or humanAPC11 and various cullins. pGBT8-PCNA and pGAD vector plasmid wereincluded as negative controls. Protein—protein interactions weredetermined by the yeast two-hybrid assay as described herein. FIG. 4Cand FIG. 4D illustrate the interaction between ROC2, APC11 and cullinfamily proteins in mammalian cells. HA-tagged ROC2 or APC11 wereco-transfected with vectors expressing CUL1 or myc-tagged individualcullin proteins into HeLa cells. Two days after transfection, cells werepulse labeled for 2 hours with [³⁵S]-methionine. Cell lysates preparedfrom the labeled cells were divided into two equal amounts,immunoprecipitated with the indicated antibodies and resolved bySDS-PAGE. All five cullin proteins were co-precipitated with HA-ROC2,but only CUL-5 co-precipitated with APC11. In the experiments shown inFIG. 4E Selective interaction between APC2 and ROC or APC11. HF7c yeastcells were co-transformed with plasmids expressing indicated proteins(key). Protein—protein interaction was determined by the yeasttwo-hybrid assay using selective medium lacking histidine (−LWH)supplemented with 5 mM 3-AT to suppress the low trans-activatingactivity of GAL4BD-APC2 fusion protein (“self-activation”).

FIGS. 5A–5F illustrates the function of ROC1 in yeast. FIG. 5Aillustrates that ScROC1 is an essential gene. Twenty tetrads from a+/roc1:kanR sporulated culture were dissected onto YPD plates, as shown.

FIG. 5B illustrates depletion of ScROC1p results in multi-budded cells.GAL-HA3-ScROC1 haploids were cultured in 2% galactose plus 2% raffinose(top panels) or 2% glucose (bottom panels) for 24 hours. DNA was stainedusing Hoechst dye.

FIG. 5C illustrates depletion of ScROC1p. GAL-HA3-ScROC1 yeast cellswere grown in either 2%, 0.05% galactose plus 2% raffinose or 2% glucosefor different length of time as indicated. Cell lysates were resolved onan SDS-PAGE gel, transferred to nitrocellulose and blotted with anti-HAantibody to detect HA3-ROC1.

FIG. 5D illustrates that ScROC1 interacts with all yeast cullins. HF7ccells (his3-200, leu2-3, trp1-901, GAL4-lacZ, GAL1-HIS3) wereco-transformed with plasmids expressing indicated proteins (key).Protein-protein interactions were determined by the yeast two-hybridassay as described in FIG. 1A.

FIG. 5E shows that human ROC1 and human ROC2 can rescue the multibuddedphenotype resulting from ScROC1p deletion. GAL-HA3-ScROC1 haploids weretransformed with pADH-414 vector, pADH-414-ScROC1, pADH-ScAPC11,pADH-hROC1 or pADH-hROC2. Transformants were streaked onto selectiveplates containing 2% glucose and grown for 24 hours when the yeast cellsdemonstrate a multiple elongated phenotype. Cells were formaldehydefixed before photography.

FIG. 5F illustrates that Sic 1 p accumulates in yeast depleted ofScROC1p. GAL-HA3-ScROC1/SIC1-HA3 yeast cells were grown in either 0.05%galactose or 2% glucose for different length of time as indicated. Celllysates were resolved on an SDS-PAGE gel, transferred to nitrocelluloseand blotted with anti-HA antibody to detect Sic1-HA3 and with anti-actinantibody to detect action to verify equal protein loading.

FIGS. 6A–6C illustrates that ROC1 stimulates cullin-dependent ubiquitinligase activity. FIG. 6A illustrates that lysates (1 mg of totalproteins) from human 293T cells transiently transfected with plasmidsexpressing indicated proteins were mixed with protein A beads linked toanti-HA antibodies. HA-immunocomplexes immobilized on the beads werewashed and then mixed with purified E1, E2 CD34 (unless otherwiseidicated), ³²P-labeled ubiquitin (ub) and ATP. After 30 minutesincubation (unless otherwise specified) at 37° C., the reactions wereterminated by boiling the samples in the presence of SDS and DTT andmixtures were resolved by SDS-PAGE, followed by autoradiography. FIG. 6Billustrates that ubiquitin ligase activity was assayed as in (A) usinglysates derived from cells transfected with plasmids expressingdifferent combination of proteins as indicated. FIG. 6C illustrates invivo ubiquitin ligase activity. Lysates from un-transfected human HeLaor 293T cells were immunoprecipated with antibodies to either ROC1,APC11 or CUL1 as indicated with (lane 4) or without competing peptide.Ubiquitin ligase activity was assayed as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which preferred embodiments ofthe invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. The amino and carboxy groups arenot presented in the sequence. Nucleotide sequences are presented hereinby single strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three letter code, in accordance with 37. C.F.R§1.822 and established usage. See, e.g., Patent In User Manual, 99–102(November 1990) (U.S. Patent and Trademark Office).

ROC1 and ROC2 (referred to herein as the “ROC proteins”), as usedherein, refer to the amino acid sequences of substantially purified ROC1and ROC2 obtained from any species, particularly mammalian, includingbovine, ovine, porcine, murine, equine, and preferably human, from anysource whether natural, synthetic, semi-synthetic, or recombinant.

An “allele” or “allelic sequence,” as used herein, is an alternativeform of the genes encoding ROC1 and ROC2. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made Up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homophenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

“Amino acid sequence,” as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of ROC1 and/orROC2 are preferably about 5 to about 15 amino acids in length and retainthe biological activity or the immunological activity of ROC1 and/orROC2. Where “amino acid sequence” is recited herein to refer to an aminoacid sequence of a naturally occurring protein molecule, amino acidsequence, and like terms, are not meant to limit the amino acid sequenceto the complete, native amino acid sequence associated with the recitedprotein molecule.

“Amplification”, as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)2, and Fc, which are capable ofbinding the epitopic determinant. Antibodies that bind ROC1 and/or ROC2polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands.

A “deletion”, as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to ROC1 and/orROC2 or the encoded ROC1 and/or ROC2. Such modifications include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Anucleic acid derivative encodes a polypeptide which retains thebiological or immunological function of the natural molecule. Aderivative polypeptide is one which is modified by glycosylation,pegylation, or any similar process which retains the biological orimmunological function of the polypeptide from which it was derived.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing. The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl,et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. AcidsRes., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al., ChemicaScripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic AcidsRes., 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch,” Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research,” Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169–176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.In addition, mixtures of naturally occurring nucleic acids and analogscan be made. Alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. “Fragments” are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding ROC1and/or ROC2, or fragments thereof, or ROC1 and/or ROC2 itself maycomprise a bodily fluid, extract from a cell, chromosome, organelle, ormembrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA (insolution or bound to a solid support, a tissue, a tissue print, and thelike). The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transfomation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transfomed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

Polynucleotides of the present invention include those coding forproteins homologous to, and having essentially the same biologicalproperties as, the proteins disclosed herein, and particularly the DNAdisclosed herein as SEQ ID NO:1 and encoding the protein ROC1 givenherein SEQ ID NO:2; as well as the DNA disclosed herein as SEQ ID NO: 3and encoding the protein ROC2 given herein as SEQ ID NO:4. Thisdefinition is intended to encompass natural allelic sequences thereof.Thus, isolated DNA or cloned genes of the present invention can be ofany species of origin, including mouse, rat, rabbit, cat, porcine, andhuman, but are preferably of mammalian origin. Thus, polynucleotidesthat hybridize to DNA disclosed herein as SEQ ID NO:1 (or fragments orderivatives thereof which serve as hybridization probes as discussedbelow) and which code on expression for a protein of the presentinvention (e.g., a protein according to SEQ ID NO:2); andpolynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:3(or fragments or derivatives thereof which serve as hybridization probesas discussed below) and which code on expression for a protein of thepresent invention (e.g., a protein according to SEQ ID NO:4), are alsoan aspect of the invention. Conditions which will permit otherpolynucleotides that code on expression for a protein of the presentinvention to hybridize to the DNA of SEQ ID NO:1 or SEQ ID NO: 3disclosed herein can be determined in accordance with known techniques.For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35–40%Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40–45% Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% Formamide with 5× Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to DNA of SEQ IDNO:1 or SEQ ID NO: 3 disclosed herein in a standard hybridization assay.See, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual(2d Ed. 1989) (Cold Spring Harbor Laboratory). In general, sequenceswhich code for proteins of the present invention and which hybridize tothe DNA of SEQ ID NO:1 or SEQ ID NO: 3 disclosed herein will be at least75% homologous, 85% homologous, and even 95% homologous or more with SEQID NO:1 or SEQ ID NO:3, respectively. Further, polynucleotides that codefor proteins of the present invention, or polynucleotides that hybridizeto that as SEQ ID NO:1 or SEQ ID NO:3, but which differ in codonsequence from SEQ ID NO:1 or SEQ ID NO:3 due to the degeneracy of thegenetic code, are also an aspect of this invention. The degeneracy ofthe genetic code, which allows different nucleic acid sequences to codefor the same protein or peptide, is well known in the literature. See,e.g., U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2, Table 1.

Although nucleotide sequences which encode ROC1 and/or ROC2 and itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring ROC1 and/or ROC2 under appropriatelyselected conditions of stringency, it may be advantageous to producenucleotide sequences encoding ROC1 and/or ROC2 or its derivativespossessing a substantially different codon usage. Codons may be selectedto increase the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encoding ROC1and/or ROC2 and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

In one embodiment of the invention, ROC nucleic acids (defined aspolynucleotides encoding ROC proteins of fragments thereof), or ROCproteins (as defined above) are initially identified by substantialnucleic acid and/or amino acid sequence identity or similarity to thesequence(s) provided herein. In a preferred embodiment, ROC nucleicacids or ROC proteins have sequence identity or similarity to thesequences provided herein as described below and one or more of the ROCprotein bioactivities as further described herein. Such sequenceidentity or similarity can be based upon the overall nucleic acid oramino acid sequence.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biol. 48,443 (1970), by the search for similarity method of Pearson &Lipman, Proc. Natl. Acad. Sci. USA 85,2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.), the Best Fit sequence program describedby Devereux et al., Nucl. Acid Res. 12, 387–395 (1984), preferably usingthe default settings, or by inspection. Preferably, percent identity iscalculated by FastDB based upon the following parameters: mismatchpenalty of 1; gap penalty of 1; gap size penalty of 0.33; and joiningpenalty of 30, “Current Methods in Sequence Comparison and Analysis,”Macromolecule Sequencing and Synthesis, Selected Methods andApplications, pp 127–149 (1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35, 351–360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5, 151–153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403–410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90, 5873–5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program which was obtained fromAltschul et al., Methods in Enzymology, 266, 460–480 (1996). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25, 3389–3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A percentage amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the coding sequenceof the cell cycle protein. A preferred method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequences in theFigures, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that shown in theFigure, as discussed below, will be determined using the number of aminoacids in the shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode ROC1 and/or ROC2 and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding ROC1 and/or ROC2 or any fragment thereof.

Knowledge of the nucleotide sequence as disclosed herein in SEQ ID NO:1or SEQ ID NO:3 can be used to generate hybridization probes whichspecifically bind to the DNA of the present invention or to mRNA todetermine the presence of amplification or overexpression of theproteins of the present invention.

The production of cloned genes, recombinant DNA, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. atCol. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling atCol. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 toWallner at Col. 6 line 8 to Col. 8 line 59. (Applicant specificallyintends that the disclosure of all patent references cited herein beincorporated herein in their entirety by reference).

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding ROC1 and/or ROC2 may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2, 318–322). In particular, genomic DNA is first amplified inthe presence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

A vector is a replicable DNA construct. Vectors are used herein eitherto amplify DNA encoding the proteins of the present invention or toexpress the proteins of the present invention. An expression vector is areplicable DNA construct in which a DNA sequence encoding the proteinsof the present invention is operably linked to suitable controlsequences capable of effecting the expression of proteins of the presentinvention in a suitable host. The need for such control sequences willvary depending upon the host selected and the transformation methodchosen. Generally, control sequences include a transcriptional promoter,an optional operator sequence to control transcription, a sequenceencoding suitable mRNA ribosomal binding sites, and sequences whichcontrol the termination of transcription and translation. Amplificationvectors do not require expression control domains. All that is needed isthe ability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants.

Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus),phage, retroviruses and integratable DNA fragments (i.e., fragmentsintegratable into the host genome by recombination). The vectorreplicates and functions independently of the host genome, or may, insome instances, integrate into the genome itself. Expression vectorsshould contain a promoter and RNA binding sites which are operablylinked to the gene to be expressed and are operable in the hostorganism.

DNA regions are operably linked or operably associated when they arefunctionally related to each other. For example, a promoter is operablylinked to a coding sequence if it controls the transcription of thesequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation. Generally,operably linked means contiguous and, in the case of leader sequences,contiguous and in reading phase.

Transformed host cells are cells which have been transformed ortransfected with vectors containing DNA coding for proteins of thepresent invention need not express protein.

Suitable host cells include prokaryotes, yeast cells, or highereukaryotic organism cells. Prokaryote host cells include gram negativeor gram positive organisms, for example Escherichia coli (E. coli) orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Exemplary host cells are E. coliW3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294(ATCC 31,446). A broad variety of suitable prokaryotic and microbialvectors are available. E. coli is typically transformed using pBR322.See Bolivar et al., Gene 2, 95 (1977). Promoters most commonly used inrecombinant microbial expression vectors include the beta-lactamase(penicillinase) and lactose promoter systems (Chang et al., Nature. 275,615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al.,Proc. Natl. Acad. Sci. USA 80, 21 (1983). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA of the present invention, i.e., they are positioned soas to promote transcription of the messenger RNA from the DNA.

Expression vectors should contain a promoter which is recognized by thehost organism. This generally means a promoter obtained from theintended host. Promoters most commonly used in recombinant microbialexpression vectors include the beta-lactamase (penicillinase) andlactose promoter systems (Chang et al., Nature 275, 615 (1978); andGoeddel et al., Nature 281, 544 (1979), a tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App.Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl.Acad. Sci. USA 80, 21 (1983). While these are commonly used, othermicrobial promoters are suitable. Details concerning nucleotidesequences of many have been published, enabling a skilled worker tooperably ligate them to DNA encoding the protein in plasmid or viralvectors (Siebenlist et al., Cell 20, 269 (1980). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA encoding the desired protein, i.e., they arepositioned so as to promote transcription of the protein messenger RNAfrom the DNA.

Eukaryotic microbes such as yeast cultures may be transformed withsuitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or anautonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979);Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157(1980). This plasmid contains the trp1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12(1977). The presence of the trp1 lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900(1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, including insect cells. Propagation of such cellsin cell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located upstream from the gene to be expressed,along with a ribosome binding site, RNA splice site (ifintron-containing genomic DNA is used), a polyadenylation site, and atranscriptional termination sequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat.No. 4,599,308. The early and late promoters are useful because both areobtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. See Fiers et al., Nature 273, 113(1978). Further, the protein promoter, control and/or signal sequences,may also be used, provided such control sequences are compatible withthe host cell chosen.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperdacells) and expression vectors such as the baculorivus expression vector(e.g., vectors derived from Autographa californica MNPV, Trichoplusia niMNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to makeproteins useful in carrying out the present invention, as described inU.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, abaculovirus expression vector comprises a baculovirus genome containingthe gene to be expressed inserted into the polyhedrin gene at a positionranging from the polyhedrin transcriptional start signal to the ATGstart site and under the transcriptional control of a baculoviruspolyhedrin promoter.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding ROC1 and/or ROC2 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing ROC1 and/or ROC2 in infected host cells(Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655–3659). Inaddition, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

Rather than using vectors which contain viral origins of replication,one can transform mammalian cells by the method of cotransformation witha selectable marker and the chimeric protein DNA. An example of asuitable selectable marker is dihydrofolate reductase (DHFR) orthymidine kinase. See U.S. Pat. No. 4,399,216. Such markers areproteins, generally enzymes, that enable the identification oftransformant cells, i.e., cells which are competent to take up exogenousDNA. Generally, identification is by survival or transformants inculture medium that is toxic, or from which the cells cannot obtaincritical nutrition without having taken up the marker protein.

In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofpeptides of the present invention without unduly adversely affecting theactivity thereof. Thus, peptides containing such deletions orsubstitutions are a further aspect of the present invention. In peptidescontaining substitutions or replacements of amino acids, one or moreamino acids of a peptide sequence may be replaced by one or more otheramino acids wherein such replacement does not affect the function ofthat sequence. Such changes can be guided by known similarities betweenamino acids in physical features such as charge density,hydrophobicity/hydrophilicity, size and configuration, so that aminoacids are substituted with other amino acids having essentially the samefunctional properties. For example: Ala may be replaced with Val or Ser;Val may be replaced with Ala, Leu, Met, or Ile, preferably Ala or Leu;Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly maybe replaced with Pro or Cys, preferably Pro; Pro may be replaced withGly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replacedwith Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replacedwith Pro or Cys, preferably Cys; His may be replaced with Phe or Gln,preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferablyHis or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe orTip; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may bereplaced with Gln or Ser, preferably Gln; KGln may be replaced with His,Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced withGln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser,preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replacedwith Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced withLys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced withArg or Asp, preferably Asp. Once made, changes can be routinely screenedto determine their effects on function with enzymes.

As noted above, the present invention provides isolated and purifiedROC1 and ROC2 proteins, such as mammalian (or more preferably human)ROC1 and ROC2. Such proteins can be purified from host cells whichexpress the same, in accordance with known techniques, or evenmanufactured synthetically.

Nucleic acids of the present invention, constructs containing the sameand host cells that express the encoded proteins are useful for makingproteins of the present invention.

Proteins of the present invention are useful as immunogens for makingantibodies as described herein, and these antibodies and proteinsprovide a “specific binding pair.” Such specific binding pairs areuseful as components of a variety of immunoassays and purificationtechniques, as is known in the alt.

The proteins of the present invention are of known amino acid sequenceas disclosed herein, and hence are useful as molecular weight markers indetermining the molecular weights of proteins of unknown structure.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding ROC1 and/or ROC2. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding ROC1 and/or ROC2, its initiation codon, and upstreamsequences are inserted into the appropriate expression vector, noadditional transcriptional or translational control signals may beneeded. However, in cases where only coding sequence, or a fragmentthereof, is inserted, exogenous translational control signals includingthe ATG initiation codon should be provided. Furthermore, the initiationcodon should be in the correct reading frame to ensure translation ofthe entire insert. Exogenous translational elements and initiationcodons may be of various origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of enhancerswhich are appropriate for the particular cell system which is used, suchas those described in the literature (Scharf, D. et al. (1994) ResultsProbl. Cell Differ. 20:125–162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; 10801 University Boulevard, Manassas, Va.20110-2209) and may be chosen to ensure the correct modification andprocessing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressROC1 and/or ROC2 may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1–2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223–32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817–23) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567–70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1–14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047–51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121–131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding ROC1 and/orROC2 is inserted within a marker gene sequence, transformed cellscontaining sequences encoding ROC1 and/or ROC2 can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding ROC1 and/or ROC2 under thecontrol of a single promoter. Expression of the marker gene in responseto induction or selection usually indicates expression of the tandemgene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding ROC1 and/or ROC2 and express ROC1 and/or ROC2 may be identifiedby a variety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridizations and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein.

The presence of polynucleotide sequences encoding ROC1 and/or ROC2 canbe detected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding ROC1 and/orROC2. Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding ROC1and/or ROC2 to detect transformants containing DNA or RNA encoding ROC1and/or ROC2.

A variety of protocols for detecting and measuring the expression ofROC1 and/or ROC2, using either polyclonal or monoclonal antibodiesspecific for the protein are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on ROC1 and/or ROC2 is preferred, but acompetitive binding assay may be employed. These and other assays aredescribed, among other places, in Hampton, R. et al. (1990; SerologicalMethods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D.E. et al. (1983; J. Exp. Med. 158:1211–1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding ROC1 and/or ROC2include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding ROC1 and/or ROC2, or any fragments thereof may be cloned into avector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and may be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3, or SP6 and labeled nucleotides. These procedures may be conductedusing a variety of commercially available kits (Pharmacia & Upjohn,(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp.,Cleveland, Ohio)). Suitable reporter molecules or labels, which may beused for ease of detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding ROC1 and/orROC2 may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode ROC1 and/or ROC2 may be designed to contain signal sequenceswhich direct secretion of ROC1 and/or ROC2 through a prokaryotic oreukaryotic cell membrane. Other constructions may be used to joinsequences encoding ROC1 and/or ROC2 to nucleotide sequence encoding apolypeptide domain which will facilitate purification of solubleproteins. Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp., Seattle, Wash.). The inclusion of cleavable linker sequences suchas those specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and ROC1 and/or ROC2 may be usedto facilitate purification. One such expression vector provides forexpression of a fusion protein containing ROC1 and/or ROC2 and a nucleicacid encoding 6 histidine residues preceding a thioredoxin or anenterokinase cleavage site. The histidine residues facilitatepurification on IMAC (immobilized metal ion affinity chromatography) asdescribed in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263–281)while the enterokinase cleavage site provides a means for purifying ROC1and/or ROC2 from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441–453).

In addition to recombinant production, fragments of ROC1 and/or ROC2 maybe produced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85, 2149–2154). Proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments ofROC1 and/or ROC2 may be chemically synthesized separately and combinedusing chemical methods to produce the full length molecule.

Antibodies that specifically bind to the proteins of the presentinvention (i.e., antibodies which bind to a single antigenic site orepitope on the proteins) are useful for a variety of diagnosticpurposes.

Antibodies to ROC1 and/or ROC2 may be generated using methods that arewell known in the art. Such antibodies may include, but are not limitedto, polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith ROC1 and/or ROC2 or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to ROC1 and/or ROC2 have an amino acid sequenceconsisting of at least five amino acids and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of ROC1 and/or ROC2 amino acids may be fused with thoseof another protein such as keyhole limpet hemocyanin and antibodyproduced against the chimeric molecule.

Monoclonal antibodies to ROC1 and/or ROC2 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. See, e.g., Kohler, G. et al. (1975) Nature,256, 495–497; Kozbor, D. et al. (1985) J. Immunol. Methods 81, 31–42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026–2030;Cole, S. P. et al. (1984) Mol. Cell Biol. 62,109–120.

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81, 6851–6855; Neuberger, M. S. et al. (1984) Nature312:604–608; Takeda, S. et al. (1985) Nature 314:452–454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceROC1 and/or ROC2-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobinlibraries (Burton D. R. (1991) Proc. Natl. Acad. Sci. 88, 11120–3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature. See,e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci, 86, 3833–3837;Winter, G. et al. (1991) Nature 349: 293–299.

Antibody fragments which contain specific binding sites for ROC1 and/orROC2 may also be generated. For example, such fragments include, but arenot limited to, the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity. See Huse, W. D. et al. (1989) Science 254,1275–1281.

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between ROC1 and/or ROC2 and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering ROC1 and/or ROC2 epitopes is preferred,but a competitive binding assay may also be employed (Maddox, supra).

Antibodies may be conjugated to a solid support suitable for adiagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, such as precipitation. Antibodies may likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescent labels (e.g., fluorescein) in accordance with knowntechniques.

Kits for determining if a sample contains proteins of the presentinvention will include at least one reagent specific for detecting thepresence or absence of the protein. Diagnostic kits for carrying outantibody assays may be produced in a number of ways. In one embodiment,the diagnostic kit comprises (a) an antibody which binds proteins of thepresent invention conjugated to a solid support and (b) a secondantibody which binds proteins of the present invention conjugated to adetectable group. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme substrates), agents forreducing background interference in a test, control reagents, apparatusfor conducting a test, and the like. A second embodiment of a test kitcomprises (a) an antibody as above, and (b) a specific binding partnerfor the antibody conjugated to a detectable group. Ancillary agents asdescribed above may likewise be included. The test kit may be packagedin any suitable manner, typically with all elements in a singlecontainer along with a sheet of printed instructions for carrying outthe test.

Assays for detecting the polynucleotides encoding ROC1 or ROC2 in acell, or the extent of amplification thereof, typically involve, first,contacting the cells or extracts of the cells containing nucleic acidstherefrom with an oligonucleotide that specifically binds to ROC1 orROC2 polynucleotide as given herein (typically under conditions thatpermit access of the oligonucleotide to intracellular material), andthen detecting the presence or absence of binding of the oligonucleotidethereto. Again, any suitable assay format may be employed (see, e.g.,U.S. Pat. No. 4,358,535 to Falkow et al.; U.S. Pat. No. 4,302,204 toWahl et al.; U.S. Pat. No. 4,994,373 to Stavrianopoulos et al; U.S. Pat.No. 4,486,539 to Ranki et al.; U.S. Pat. No. 4,563,419 to Ranki et al.;and U.S. Pat. No. 4,868,104 to Kurn et al.) (the disclosures of whichapplicant specifically intends be incorporated herein by reference).

Antisense oligonucleotides and nucleic acids that express the same maybe made in accordance with conventional techniques. See, e.g., U.S. Pat.No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. Thelength of the antisense oligonucleotide (i.e., the number of nucleotidestherein) is not critical so long as it binds selectively to the intendedlocation, and can be determined in accordance with routine procedures.In general, the antisense oligonucleotide will be from 8, 10 or 12nucleotides in length up to 20, 30, or 50 nucleotides in length. Suchantisense oligonucleotides may be oligonucleotides wherein at least one,or all, or the internucleotide bridging phosphate residues are modifiedphosphates, such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues may be modified as described. In another non-limiting example,such antisense oligonucleotides are oligonucleotides wherein at leastone, or all, of the nucleotides contain a 2′ loweralkyl moiety (e.g.,C₁–C₄, linear or branched, saturated or unsaturated alkyl, such asmethyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).For example, every other one of the nucleotides may be modified asdescribed. See also P. Furdon et al., Nucleic Acids Res. 17, 9193–9204(1989); S. Agrawal et al., Proc. Natl. Acad. Sci. USA 87,1401–1405(1990); C. Baker et al., Nucleic Acids Res. 18, 3537–3543 (1990); B.Sproat et al., Nucleic Acids Res. 17, 3373–3386 (1989); R. Walder and J.Walder, Proc. Nat. Acad. Sci. USA 85, 5011–5015 (1988).

In a preferred embodiment, the ROC proteins, nucleic acids, variants,modified proteins, cells and/or transgenics containing the ROC nucleicacids or proteins are used in screening assays. Identification of theROC proteins provided herein permits the design of drug screening assaysfor compounds that bind or interfere with the binding to the ROCproteins and for compounds which modulate ROC activity.

The assays described herein preferably utilize the human ROC proteins,although other mammalian proteins may also be used, including rodents(mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep,pigs, horses, etc.) and primates. These latter embodiments may bepreferred in the development of animal models of human disease. In someembodiments, as outlined herein, valiant or derivative ROC proteins maybe used, including deletion ROC proteins as outlined above.

In a preferred embodiment, the methods comprise combining a ROC proteinand a candidate bioactive agent, and determining the binding of thecandidate agent to the ROC proteins. In other embodiments, furtherdiscussed below, binding interference or bioactivity is determined.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, small organic molecule,carbohydrates (including polysaccharides), polynucleotide, lipids, etc.Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls, i.e. the use of agents knownto alter ROC activity, may be used.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, a library of different candidate bioactiveagents are used. Preferably, the library should provide a sufficientlystructurally diverse population of randomized agents to effect aprobabilistically sufficient range of diversity to allow binding to aparticular target. Accordingly, an interaction library should be largeenough so that at least one of its members will have a structure thatgives it affinity for the target. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷–10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for the target. Forexample, a library of all combinations of a peptide 7 to 20 amino acidsin length, has the potential to code for 20⁷ (10⁹) to 20²⁰. Thus, withlibraries of 10⁷ to 10⁸ different molecules the present methods allow a“working” subset of a theoretically complete interaction library for 7amino acids, and a subset of shapes for the 20²⁰ library. Thus, in apreferred embodiment, at least 10⁶, preferably at least 10⁷, morepreferably at least 10⁸ and most preferably at least 10⁹ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

In a preferred embodiment, the candidate bioactive agents are proteins.In another preferred embodiment, the candidate bioactive agents arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, may beused. In this way libraries of prokaryotic and eukaryotic proteins maybe made for screening in the systems described herein. Particularlypreferred in this embodiment are libraries of bacterial, fungal, viral,and mammalian proteins, with the latter being preferred, and humanproteins being especially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. In another preferred embodiment, the candidate bioactive agentsare organic chemical moieties, a wide variety of which are available inthe literature.

In one embodiment of the methods described herein, portions of ROCproteins are utilized; in a preferred embodiment, portions having ROCactivity are used. ROC activity is as described herein and includesbinding activity to cullins as outlined herein. In addition, the assaysdescribed herein may utilize either isolated ROC proteins or cellscomprising the ROC proteins.

Generally, in a preferred embodiment of the methods herein, for examplefor binding assays, the ROC proteins or the candidate agent isnon-diffusibly bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can bebound, is readily separated from soluble material, and is otherwisecompatible with the overall method of screening. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose, TEFLON®, etc.Microtiter plates and arrays are especially convenient because a largenumber of assays can be carried out simultaneously, using small amountsof reagents and samples. In some cases magnetic beads and the like areincluded. The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the composition andis nondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block important sites on the proteinwhen the protein is bound to the support), direct binding to “sticky” orionic supports, chemical crosslinking, the synthesis of the protein oragent on the surface, etc. Following binding of the protein or agent,excess unbound material is removed by washing. The sample receivingareas may then be blocked through incubation with bovine serum albumin(BSA), casein or other innocuous protein or other moiety. Also includedin this invention are screening assays wherein solid supports are notused; examples of such are described below.

In a preferred embodiment, the ROC proteins is bound to the support, anda candidate bioactive agent is added to the assay. Alternatively, thecandidate agent is bound to the support and the ROC proteins is added.Novel binding agents include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of particular interest are screening assays for agents that have alow toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein—protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays, and the like.

The determination of the binding of the candidate bioactive agent to theROC proteins may be done in a number of ways. In a preferred embodiment,the candidate bioactive agent is labelled, and binding determineddirectly. For example, this may be done by attaching all or a portion ofthe ROC proteins to a solid support, adding a labelled candidate agent(for example a fluorescent label), washing off excess reagent, anddetermining whether the label is present on the solid support. Variousblocking and washing steps may be utilized as is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures. The label can directly or indirectly provide a detectablesignal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. ROC proteins), such as an antibody, peptide,binding partner, ligand, etc. In a preferred embodiment, the competitoris a cullin. Under certain circumstances, there may be competitivebinding as between the bioactive agent and the binding moiety, with thebinding moiety displacing the bioactive agent. This assay can be used todetermine candidate agents which interfere with binding between ROCproteins and its biological binding partners. “Interference of binding”as used herein means that native binding of the ROC proteins differs inthe presence of the candidate agent. The binding can be eliminated orcan be with a reduced affinity. Therefore, in one embodiment,interference is caused by, for example, a conformation change, ratherthan direct competition for the native binding site.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40 C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the ROCproteins and thus is capable of binding to, and potentially modulating,the activity of the ROC proteins. In this embodiment, either componentcan be labeled. Thus, for example, if the competitor is labeled, thepresence of label in the wash solution indicates displacement by theagent. Alternatively, if the candidate bioactive agent is labeled, thepresence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the ROC proteins with a higher affinity. Thus, if thecandidate bioactive agent is labeled, the presence of the label on thesupport, coupled with a lack of competitor binding, may indicate thatthe candidate agent is capable of binding to the ROC proteins.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating the activityof the ROC proteins. Such assays can be done with the ROC proteins orcells comprising said ROC proteins. In one embodiment, the methodscomprise combining an ROC proteins and a competitor in a first sample. Asecond sample comprises a candidate bioactive agent, an ROC proteins anda competitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the ROCproteins and potentially modulating its activity. That is, if thebinding of the competitor is different in the second sample relative tothe first sample, the agent is capable of binding to the ROC proteins.

Alternatively, a preferred embodiment utilizes differential screening toidentify drug candidates that bind to the native ROC proteins, butcannot bind to modified ROC proteins. The structure of the ROC proteinsmay be modeled, and used in rational drug design to synthesize agentsthat interact with that site. Drug candidates that affect cell cyclebioactivity are also identified by screening drugs for the ability toeither enhance or reduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein—proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Screening for agents that modulate the activity of the ROC proteins mayalso be done. In a preferred embodiment, methods for screening for abioactive agent capable of modulating the activity of ROC proteinscomprise the steps of adding a candidate bioactive agent to a sample ofa ROC proteins (or cells comprising a ROC proteins) and determining analteration in the biological activity of the ROC proteins. “Modulatingthe activity of a ROC proteins” includes an increase in activity, adecrease in activity, or a change in the type or kind of activitypresent. Thus, in this embodiment, the candidate agent should both bindto the ROC protein (although this may not be necessary), and alter itsbiological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods, as are generally outlinedabove, and in vivo screening of cells for alterations in the presence,distribution, activity or amount of ROC proteins.

Thus, in this embodiment, the methods comprise combining a ROC proteinand a candidate bioactive agent, and evaluating the effect on thebioactivity of the ROC proteins. By “ROC protein activity” orgrammatical equivalents herein is meant at least one of the ROCproteins' biological activities, including, but not limited to, theproteins' ability to bind cullins (including, but not limited to, cullin1, 2, 3, 4A and 5), its activity in ligating ubiquitin and theubiquitin-dependent proteolytic process, its role in SICp degradation,and any other activity of ROC proteins as described herein, etc.

In a preferred embodiment, the activity of the ROC proteins isdecreased; in another preferred embodiment, the activity of the ROCproteins is increased. Thus, bioactive agents that are antagonists arepreferred in some embodiments, and bioactive agents that are agonistsmay be preferred in other embodiments.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of an ROCproteins. The methods comprise adding a candidate bioactive agent, asdefined above, to a cell comprising ROC proteins. Preferred cell typesinclude almost any cell. The cells contain a recombinant nucleic acidthat encodes a ROC protein. In a preferred embodiment, a library ofcandidate agents are tested on a plurality of cells.

Detection of ROC activity may be done as will be appreciated by those inthe art. There are a number of parameters that may be evaluated orassayed to allow the detection of alterations in ROC bioactivity.

The measurements can be determined wherein all of the conditions are thesame for each measurement, or under various conditions, with or withoutbioactive agents, etc. For example, measurements of ROC activity can bedetermined in a cell or cell population wherein a candidate bioactiveagent is present and wherein the candidate bioactive agent is absent. Inanother example, the measurements of ROC activity are determined whereinthe condition or environment of the cell or populations of cells differfrom one another. For example, the cells may be evaluated in thepresence or absence or previous or subsequent exposure of physiologicalsignals, for example hormones, antibodies, peptides, antigens,cytokines, growth factors, action potentials, pharmacological agentsincluding chemotherapeutics, radiation, carcinogenics, or other cells(i.e. cell—cell contacts).

By a “population of cells” or “library of cells” herein is meant atleast two cells, with at least about 10³ being preferred, at least about10⁶ being particularly preferred, and at least about 10⁸ to 10⁹ beingespecially preferred. The population or sample can contain a mixture ofdifferent cell types from either primary or secondary cultures althoughsamples containing only a single cell type are preferred, for example,the sample can be from a cell line, particularly tumor cell lines. In apreferred embodiment, cells that are replicating or proliferating areused; this may allow the use of retroviral vectors for the introductionof candidate bioactive agents. Alternatively, non-replicating cells maybe used, and other vectors (such as adenovirus and lentivirus vectors)can be used. In addition, although not required, the cells arecompatible with dyes and antibodies.

Preferred cell types for use in the invention include, but are notlimited to, mammalian cells, including animal (rodents, including mice,rats, hamsters and gerbils), primates, and human cells, particularlyincluding tumor cells of all types, including breast, skin, lung,cervix, colonrectal, leukemia, brain, etc.

The proteins and nucleic acids provided herein can also be used forscreening purposes wherein the protein—protein interactions of the ROCproteins can be identified. Genetic systems have been described todetect protein—protein interactions. The first work was done in yeastsystems, namely the “yeast two-hybrid” system. The basic system requiresa protein—protein interaction in order to turn on transcription of areporter gene. Subsequent work was done in mammalian cells. See Fieldset al., Nature 340, 245 (1989); Vasavada et al., Proc. Natl. Acad. Sci.USA 88, 10686 (1991); Fearon et al., Proc. Natl. Acad. Sci. USA 89, 7958(1992); Dang et al., Mol. Cell. Biol, 11, 954 (1991); Chien et al.,Proc. Natl. Acad. Sci. USA 88, 9578 (1991); and U.S. Pat. Nos.5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463.

In general, two nucleic acids are transformed into a cell, where one isa “bait” such as the gene encoding a ROC proteins or a portion thereof,and the other encodes a test candidate. Only if the two expressionproducts bind to one another will an indicator, such as a fluorescentprotein, be expressed. Expression of the indicator indicates when a testcandidate binds to the ROC proteins. Using the same system and thenewly-identified proteins the reverse can be performed. Namely, the ROCproteins provided herein can be used to identify new baits, or agentswhich interact with ROC proteins. Additionally, the two-hybrid systemcan be used wherein a test candidate is added in addition to the baitand the ROC proteins encoding nucleic acids to determine agents whichinterfere with the bait, such as cullins.

In this way, bioactive agents are identified. Bioactive agents (i.e.,compounds) with pharmacological activity are those compounds that areable to enhance or interfere with the activity of at least one of theROC proteins. The compounds having the desired pharmacological activitymay be administered in a pharmaceutically acceptable carrier (i.e., apharmaceutical formulation) to a host or subject Suitable subjects arepreferably human subjects, but may also be other mammalian subjects,such as dogs, cats and livestock (i.e., for veterinary purposes).

Pharmaceutical formulations of the present invention comprise compoundswith pharmacological activity (as identified using methods of thepresent invention) in a pharmaceutically acceptable carrier. Suitablepharmaceutical formulations include those suitable for inhalation, oral,rectal, topical, (including buccal, sublingual, dermal, vaginal andintraocular), parenteral (including subcutaneous, intradermal,intramuscular, intravenous and intraarticular) and transdermaladministration. The compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart. The most suitable route of administration in any given case maydepend upon the anatomic location of the condition being treated in thesubject, the nature and severity of the condition being treated, and theparticular pharmacologically active compound which is being used. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art.

In the manufacture of a medicament according to the invention (the“formulation”), pharmacologically active compounds or thephysiologically acceptable salts thereof (the “active compounds”) aretypically admixed with, inter alia, an acceptable carrier. The carriermust, of course, be acceptable in the sense of being compatible with anyother ingredients in the formulation and must not be deleterious to thepatient. The carrier may be a solid or a liquid, or both, and ispreferably formulated with the compound as a unit-dose formulation, forexample, a tablet, which may contain from 0.5% to 99% by weight of theactive compound. One or more active compounds may be incorporated in theformulations of the invention, which formulations may be prepared by anyof the well known techniques of pharmacy consisting essentially ofadmixing the components, optionally including one or more accessorytherapeutic ingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound ofFormula (I), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe active compound.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the pharmaceuticallyactive compounds identified with the methods described herein may belyophilized to produce a lyophilizate which may be reconstituted with apharmaceutically acceptable carrier, such as water, to regenerate aliposomal suspension.

Other pharmaceutical formulations may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the formulation willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

In addition to the pharmacologically active compounds, thepharmaceutical formulations may contain other additives, such aspH-adjusting additives. In particular, useful pH-adjusting agentsinclude acids, such as hydrochloric acid, bases or buffers, such assodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodiumborate, or sodium gluconate. Further, the compositions may containmicrobial preservatives. Useful microbial preservatives includemethylparaben, propylparaben, and benzyl alcohol. The microbialpreservative is typically employed when the formulation is placed in avial designed for multidose use. Of course, as indicated, thepharmaceutical formulations of the present invention may be lyophilizedusing techniques well known in the art.

The therapeutically effective dosage of any specific pharmacologicallyactive compound identified by methods of the invention, the use of whichcompounds is in the scope of present invention, will vary somewhat fromcompound to compound, and subject to subject, and will depend upon thecondition of the patient and the route of delivery.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLE 1 Materials and Methods cDNA Clones, Plasmids Constructs andYeast Two Hybrid Assay

A cDNA sequence encoding full-length mouse cullin 4A was used as a baitto screen a HeLa cell derived cDNA library for cullin-interactingproteins by the yeast two-hybrid assay described in Michel and Xiong,Cell Growth. Differ. 9, 439–445 (1998). The full length cDNA clones forboth human ROC2 and APC11 were isolated by PCR amplification from a HeLacDNA library and confirmed by DNA sequencing. To identify cDNA clonesencoding the full length mammalian APC2, the EST database was searched.Full length cDNA clones were not available for human APC2 in the presentEST database. Instead, a near full-length mouse APC2 EST cDNA clone(W13204) was identified that predicts a 823 amino acid open readingframe with a calculated molecular weight of 94 kDa. This mouse cDNAclone is one amino acid residue longer than the published human APC2 (Yuet al., 1998a), but is missing the initiation methionine codon. Giventhe extremely close relatedness between mouse and human APC2 proteins(93% identity over the entire 823 residues), the mouse APC2 was usedwhen testing for the interaction with human APC11.

Yeast cDNA sequences were amplified from yeast genomic DNA by PCR andverified by DNA sequencing. The primers used for ScROC1 were: 5′-TTT AAAGAG AAA TAG GAT CCC ATG AGC AAC GAA-3′ [SEQ ID NO: 5] and 5′-TTA AAT GTTTAC GGG GAA TTC ATT TTT TCA CCT-3′ [SEQ ID NO: 6] incorporating a 5′BamHI site and a 3′ EcoRI site (underlined) by which the PCR product wasinserted in frame into the pGAD prey vector. pGBT8-ScROC1 wasconstructed using SmaI and SacI restriction sites from pGEX-ScROC1.Primers for amplifying ScAPC11 are: 5′-GGC AAT ACA GAT TAG GAT CCT ATGAAA GTT AAA-3′[SEQ ID NO: 7] and 5′-AAT TGT GAT TTC TAG AAT TCT TTT TTATCG TAA-3′ [SEQ ID NO: 8] incorporating a 5′ BamHI site and a 3′ EcoRIsite (underlined) by which the PCR product was inserted in frame intothe pGAD vector. CDC53 was provided by Dr. Mike Tyers and was subclonedfrom pMT1144 into pBSKS using BamHI and NotI sites. From here it wassubcloned in frame into pGBT8 using BamHI and SacI sites to createpGBT8-CDC53. CUL B (ORF YGR003w) was PCR cloned using primers: 5′-ATCCCC ATG GCT ATG ATA ACT AAT AAG AAA ATA-3′ [SEQ ID NO: 9] and 5′-CTG CAGAGC TCG TTA GGA AAG GTA ATG GTA ATA-3′ [SEQ ID NO: 10] incorporating a5′ NcoI site and a 3′ SacI site (underlined) by which the PCR productwas inserted in frame into the pGBT8 bait vector. CUL C (ORF YJL047c)was PCR cloned using primers: 5′-ATC CCC ATG GCT ATG ATA AAT GAG AGC GTTTCC-3′ [SEQ ID NO: 11] and 5′-AGC TCG TCG ACA TTA GTA CTT GTA AGT TGCTAT-3′ [SEQ ID NO: 12] incorporating a 5′ NcoI site and a 3′ Sail site(underlined) by which the PCR product was inserted in frame into thepGBT8 bait vector. ScAPC2 was PCR cloned using primers: 5′-ATC CCC ATGGCT ATG TCA TTT CAG ATT ACC CCA-3′ [SEQ ID NO: 13] and 5′-AGC TCG TCGACA TCA TGA GTT TTT ATG CCC ATT-3′ [SEQ ID NO: 14] incorporating a 5′NcoI site and a 3′ SalI site (underlined) by which the PCR product wasinserted in frame into the pGBT8 bait vector. All PCR clonings were doneusing lyticase treated YEF473 genomic DNA as template using thefollowing protocol: 1 min 94° C., 1 min 55° C., 1 min/kb 68° C. for 25cycles followed by a 10 min extension at 68° C. For ScROC1 and ScAPC11,Pfu proofreading DNA polymerase (Stratagene) was used in reactionscontaining 1×PCR buffer, 2.5 mM MgCl₂, 0.5 mM each primer and 0.1 mMdNTPs. For PCR amplification of CUL B, CUL C and ScAPC2, the longtemplate Expand kit (Boehringer Mannheim) was used followingmanufacturer's instructions. Reactions contained 0.1 mM MgCl₂ (Buffer1), 0.2 mM dNTPs, 0.5 mM each primer and 0.1 mg/ml BSA. ScROC1, ScAPC11,hROC1 and hROC2 were all inserted into the p414-ADH vector (CEN) using5′ BamHI and 3′ XhoI restriction sites.

For expression in mammalian cells, individual cDNA clones were subclonedinto the pcDNA3 vector under the control of CMV promoter (Invitrogen),pcDNA3-HA or pcDNA3-Myc, for expressing HA or myc epitope tagged fusionprotein. For the yeast two-hybrid assay, individual cullin sequenceswere cloned into pGBT8, a modified version of pGBT9, in frame with theDNA-binding domain of Gal4. ROC1, ROC2 and APC11 were cloned into pGADin-frame with the DNA activation domain of Gal4. Yeast two-hybridexpression plasmids for human CUL1, CUL1 deletion mutants and SKP1 werepreviously described (Michel and Xiong, 1998, supra).

EXAMPLE 2 Materials and Methods Cell Lines, Culture Conditions and CellTransfection

All mammalian cells were cultured in DMEM, supplemented with 10% FBS ina 37° C. incubator with 5% CO₂, which include HeLa (human cervixepithelioid carcinoma), Saos-2 (osteosarcoma), and 293T (humantransformed primary embryonal kidney c cells). Cell transfections werecarried out using the LipofectAMINE reagent according to themanufacturer's instructions (Gibco-BRL). For each transfection, 4 μg oftotal plasmid DNA (adjusted with pcDNA3 vector DNAs) was used for 60 mmdish.

EXAMPLE 3 Materials and Methods Antibodies and ImmunochemistryProcedures

Procedures for [³⁵S]-methionine metabolic labeling, immunoprecipitationand immunoblotting have been described previously (Jenkins, C. W. andXiong, Y. (1995), “Immunoprecipitation and immunoblotting in cell cyclestudies” in Cell Cycle: Material and methods, M. Pagano, ed. (New York:Springer-Verlag), pp. 250–263). The sequence of synthetic peptides usedin generating rabbit polyclonal antibodies are as follows: anti-humanROC1N (CMAAAMDVDTPSGTN, amino acid residues 1–14 [SEQ ID NO:15],anti-human ROC1C (CDNREWEFQKYGH, residues 97–108 [SEQ ID NO: 16],anti-human APC11 (CRQEWKFKE, residues 76–84) [SEQ ID NO: 17], andanti-human CUL2 [CRSQASADEYSYVA, residues 733–745 [SEQ ID NO: 18]. SeeKipreos et al., 1996, supra; Michel and Xiong, 1998, supra. A cysteine(underlined) was added to the N-terminus of each peptide for covalentcoupling of the peptide to activated keyhole limpet haemocyanin (KLH).Antibodies to human CUL1 and SKP1 were previously described (Michel andXiong, 1998, supra). All rabbit polyclonal antibodies used in this studywere affinity purified using respective peptide columns following themanufacturer's instruction (Sulfolink Kit, Pierce, Rockford, Ill.).Monoclonal anti-HA (12CA5, Boehringer-Mannheim) and anti-myc (9E10,NeoMarker) antibodies were purchased commercially. Antibody to yeastactin was provided by Dr. J. Pringle. Coupled in vitro transcription andtranslation reactions were performed using the TNT kit following themanufacturer's instructions (Promega).

EXAMPLE 4 Materials and Methods Immunopurification of ROC1 Complexes andProtein Microsequencing

For preparative scale immunopurification of ROC1 complexes, total lysatewas prepared from the HeLa cells pooled from ten 150 mm plates afterlysis with the NP-40 lysis buffer and clarified by high speedcentrifugation (13,000 g for 30 minutes). Following pre-clearing withuncoated sephadex beads, 100 μg of affinity purified antibodies to humanROC1 was added to the clarified cell lysate. After incubating at 4° C.with rotation for 1 hour, protein A beads were added to the lysate andincubated for 1 hour. The beads were washed three times with NP-40 lysisbuffer, boiled for 3 minutes in Laemmli loading buffer, and the proteinswere separated by SDS-PAGE. After silver staining, ROC1-specificassociated bands were identified by comparing with a parallelimmunoprecipitation of the same HeLa lysate with the same anti-ROC1antibody in the presence of molar excess of competing antigen peptide.Competable bands at molecular weight between 70 to 120 kDa were excisedfrom the SDS gel and subjected to in-gel protease digestion usinglysylendopeptidase (50 ng/ml). Digested peptide fragments was extractedby acetonitrile and separated by reverse-phase high pressure liquidchromatography on a Hewlett Packard 1100 HPLC system using a C18 column(1 mm×250 mun, Vydac). Protein sequences of individual peptidescollected from HPLC were determined on an automated ABI microsequencerat Glaxo-Wellcome protein microsequencing facility.

EXAMPLE 5 Materials and Methods Yeast Strains

All S. cerevisiae strains were derived from YEF473 (a/α ura3-52/ura3-52his3Δ-200/his3Δ-200 trp1Δ-63/trp1Δ-63 leu2Δ-1/leu2Δ-1lys2-801/lys2-801). Yeasts were cultured at 30° C. unless otherwiseindicated in YP medium or SD medium (lacking appropriate amino acids)containing 2% glucose or 2% raffinose plus varying amounts of galactose,as appropriate. To determine protein expression, yeast cultures werecollected by centrifugation, washed once with distill water and storedat −80° C. overnight. Cell pellets were resuspended in lysis buffercontaining 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2% Triton X-100, 1 mMDTT, 1 mM PMSF, 1 mM NaVO₃ and 1× protease inhibitors (25 μl/mlleupeptin, 25 μ/ml aprotonin, 1 mM benzamidine and 10 μ/ml tyrpsininhibitor). Glass beads were added, and samples were vortexed 4×30 secwith at least 30 sec on ice between each vortex. Suspension wastransferred to a new Eppendorf tube and centrifuged at 13000 g at 4° C.for 30 min. Protein concentration in the whole cell extract weremeasured using Broadford assay and equal amount of total protein fromeach sample was separated by SDS-PAGE and followed by immunoblotting.For nuclei staining, yeast was fixed in 3.7% formaldehyde in culturingmedium for 1 hr. in a roller drum at 30° C. Fixed cells were washed 3times in 1×PBS and resuspended in mounting medium (1% w/vp-phenylenediamine (Sigma) in 1×PBS pH 9, 90% glycerol and 0.5 μg/mlHoechst 33258 dye) as described in Pringle, J. R. et al., (1991),“Immunofluorescence methods for yeast” in Methods in Enzymol. 194,565–602.

Mutant yeast strains were constructed using PCR-based gene deletion andmodification by homologous recombination according to Longtine et al.,Yeast 14(10), 953–961 (1998). Primers for PCR products for all strainsconstructed were designed based on the sequences published in thedatabase and contained 40 bp of sequence homologous to the gene specificsequence (upper case) and 20 bp homologous to the vector template (lowercase). To create a perfect deletion of ROC1 and replace it with a modulecontaining the E. coli kanr gene (strain JM1), pFA6a-kanMX6 template wasused with primers ROC1-F1(5′-TTCTCCAGTGGCAGAGAACTTTAAAGAGAAATAGTTCAACcggatccccgggtt aa-ttaa 5′)[SEQ ID NO: 19] and ROC1-R1(5′-ACCTCGGTATGATTTAAATGTTTACGGGCAATTCATTTTTgaattc-gagctcgtttaaac 3′)[SEQ ID NO: 20]. To integrate chromosomally the S. pombe his5+ genefollowed by the GAL1 promoter and an HA3 tag in frame with the ScROC1gene (strain JM5), pFA6a-His3MX6-pGAL1-HA3 template was used withprimers ROC1-F4 (5′ATAGACGTATGGGCTTCAATAT-GTGCAATGTTGGTTGCTAgaattcgagctcgtttaaac-3′) [SEQID NO: 21] and ROC1-R3 (5′CATCTTCATCAACA-TCCATCCTGTCAACTTCGTTGCTCATgcactgagcagcgtaat-ctg3′) [SEQID NO: 22]. To epitope tag the C-terminus of SIC1 with HA3 tag followedby the TRP1 selectable marker (strain JM7), pFA6a-HA3-TRP1 template wasused with primers SIC1-F2 (5′CAAGCCAAAGGCATTGTTTCAATCTAGGGAT-CAAGAGCATcggatccccgggttaattaa 3′) [SEQID NO: 23] and SIC1-R1 (5′TAAAATATAATCGTTCCAGAAA-CTTTTTTTTTTCATTTCTgaattcgagctcgtttaaac 3′)[SEQ IDNO: 24]

PCR was performed using the Expand Long Template PCR System (BoehringerMannheim) with the following protocol. Mix 1 (25 μl) contained 2.5 μlExpand Buffer 1, 0.8 mM dNTPs, 10 μg BSA and 2 mM each primer. Mix 2(100 μl) contained 7.5 μl Expand Buffer 1, 0.75 μL Expand enzymemixture, and 0.1 μg template DNA. The two mixes were added together,mixed well and immediately subjected to PCR: 20 cycles of 1 min 94° C.,1 min 55° C., 1 min/kb 68° C. followed by a 10 min extension at 68° C.PCR products from at least eight reactions were pooled, extracted oncewith phenol:chlorofolm:isoamyl alcohol (25:24:1) and ethanolprecipitated. PCR products were transformed into diploid YEF473 yeast(to construct strains JM1 and JM5) or into the haploid strain JM5 (toconstruct strain JM7) using a standard protocol and plated onto richmedium (YPD plates for strains JM1 and JM5, and YP plates plus 2%raffinose and 2% galactose for strain JM7) for two days. Plates werethen replica plated onto appropriate selectable medium for 2–3 days.Selected transformants were streaked onto selectable medium twice. Toidentify transformants that had integrated by homologous recombination,PCR was performed on genomic DNA prepared by lyticase treatment usingone primer that annealed to the module integrated and one primer thatannealed to a region outside of that altered by the recombination. PCRproduct of the appropriate size confirmed homologous recombination. Inaddition, 2:2 segregation of the selectable marker also confirmedhomologous recombination.

EXAMPLE 6 Materials and Methods Ubiquitin Ligase Activity Assay

The detailed procedures for the purification of human E1 and mouse E2CDC34, the preparation of 32P-labeled ubiquitin, as well asimmuno-purification of the ROC1/CUL1 containing E3 ligase complex fromtransiently transfected 293T cells (FIG. 6) is described in anaccompanying paper (Tan et al.). For immunoprecipitation fromun-transfected cells, 2 μg of affinity-purified anti-ROC1C, anti-CUL1,or anti-APC11 was used. The immuno-purified ROC1/CUL1 containing compleximmobilized on protein A agarose beads was added to an ubiquitinligation reaction mixture (30 μl) that contained 50 mM Tris-HCl, pH 7.4,5 mM MgCl₂, 2 mM NaF, 10 nM Okadaic Acid, 2 mM ATP, 0.6 mM DTT, 1 μg32P-Ub, 60 ng E1 and 300 ng mouse CDC34 protein. The incubation was at37° C. for 30 min unless otherwise specified herein. The reactionmixture was then added to 20 μl 4× Laemmli loading buffer with 10 mMDDT, and boiled for 3 min prior to 7.5% SDS-PAGE analysis.

EXAMPLE 7 ROC1 Interacts Directly with All Cullins

Using the yeast two-hybrid screen for cellular proteins that interactwith the cullin family of proteins, as described above, a human HeLacDNA library was screened using mouse cullin 4A as a bait. Full lengthmouse CUL4A encodes a 759 amino acid protein and shares 96% identitywith human CUL4A that was recently identified as a candidate 13qamplicon target gene and was amplified or overexpressed in highpercentage of breast cancer samples (Chen et al., 1998, supra). Anestimated 3×10⁶ transformants were screened. Of 17 clones isolated fromthis screen that grew on histidine deficient selective medium, 11corresponded to the gene, named ROC1 (regulator of cullins), asdetermined by DNA sequencing and diagnostic restriction digestionanalysis. The DNA sequence of ROC1 is provided herein in FIG. 2A as SEQID NO:1. In addition to CUL4A, ROC1 can also interact with cullin 1, 2and 5 as determined by the yeast two-hybrid assay (FIG. 1A). Cullin 3which interacts with ROC1 very weakly in yeast cells was later found toalso bind to ROC1 in cultured mammalian cells (see below). Thus, ROC1,unlike SKP1 which selectively interacts with CULL only (cf. Michel andXiong, 1998, supra, FIG. 1A), appears to be a general cullin-interactingprotein.

The mammalian cullin genes encode a family of closely related proteinswith molecular weights of approximately 90 kDa. CUL1 interacts with SKP1via an NH₂-terminal domain (see Michel and Xiong, 1998, supra). Todetermine the structural basis underlying the specific interactionbetween cullins and ROC1, the region of CUL1 required for itsinteraction with ROC1 was mapped. A series of CUL1 deletions from bothamino- and carboxyl-terminals fused in-frame with the yeast Gal4 DNAbinding domain were tested for their abilities to interact with ROC1 inyeast cells. ROC1 interacts with the C-terminal 527 amino acid residuesof CUL1, but not the N-terminal 249 residues of CUL1 (FIG. 1B). Incontrast, SKP1 binds to the N-terminal domain of CUL1. These resultsindicate that CUL1 contains at least two distinct domains, an N-terminaldomain for interacting with SKP1 and a C-terminal domain for bindingwith ROC1. Such structural separation suggests that ROC1 is unlikely tointeract with CUL1 in a competing manner with SKP1. Hence, ROC1 and SKP1may co-exist in the same protein complex with CUL1 to perform differentfunctions.

EXAMPLE 8 ROC1 Represents a Family of RING Finger Proteins Related toAPC11

ROC1 encodes an 108 amino acid residue protein with a predictedmolecular weight of 12265 D (FIG. 2A, SEQ ID NO: 2). Database searchesidentified ROC1 as a highly evolutionarily conserved gene whose S.cerevisiae (ROC1-Sc), S. pombe (ROC1-Sp) and plant (ROC1-At) homologuesshare 67%, 88% and remarkably 98% protein sequence identity with humanROC1, respectively, over the 82 amino acid region compared (FIG. 2C).Database searches have also identified two additional genes, ROC2 inhigher eukaryotes and APC11 in all eukaryotic species (FIGS. 2B and 2C),that are closely related to ROC1. Human ROC2 and APC11 encode an 85amino acid (Mr. 10007 D) and an 84 residue (Mr. 9805 D) protein,respectively. The DNA sequence of ROC2 is provided herein at FIG. 2B asSEQ ID NO:3; its amino acid sequence is provided herein at FIG. 2B asSEQ ID NO:4. ROC1 and ROC2 share an overall protein sequence identity of51% with each other and 38% and 35% identity with APC11, respectively,indicating that ROC1 and ROC2 are more closely related to each otherthan to APC11, Like ROC1, both ROC2 and APC11 are also highly conservedduring evolution. Therefore, ROC1/ROC2/APC11 define a new family ofproteins that are likely to carry out important cellular functions.

ROC/APC11 proteins contain two characteristic features: a RING fingerand richness in tryptophan residues. The RING finger domain has beenfound in many eukaryotic proteins with diverse functions and is thoughtto mediate protein—protein interactions (Borden, K. L. and Freemont, P.S. (1996), Current Opinion in Structural Biology 6, 395–401). Themajority of RING finger proteins contain a highly conserved structuralmotif with a histidine residue flanked by three and four cysteineresidues on either side (C₃HC₄). Notably, the ROC1 protein from allspecies has a substitution of the last cysteine with an aspartic acidresidue (FIG. 2C). The second feature of this family of proteins is sixhighly conserved tryptophan residues. Three tryptophan residues in ROC1are followed by an acidic amino acid residue (Asn, Glu or Asp) thatresemble the WD repeat and may potentially also be involved in mediatingprotein—protein interactions.

APC11 was recently identified as a subunit of the yeast APC complexwhose loss of function resulted in a defect in the onset of anaphase andexit from mitosis (Zachariae et al., 1998, supra). Another APC subunit,APC2, was found to contain limited sequence similarity to the C-terminalregion of cullins. Id. Although Applicant does not wish to be bound toany theory of the invention, these observations, together with thefinding that both ROC1 and ROC2 (see below) directly interact withcullins, suggest (1) that APC11 may directly interact with APC2, (2)that the region for interacting with ROC and APC11 may be located in theconserved C-terminal portion in cullin and APC2 proteins, and (3) thatROC proteins may function in regulating ubiquitin-dependent proteolysis.

EXAMPLE 9 In Vivo Association of ROC1 and Cullins

To confirm the interaction between ROC1 and cullin proteins, Saos-2cells were transfected with plasmids directing the expression ofHA-epitope tagged human ROC1 (HA-ROC1) together with CUL1 or otherindividual myc-epitope tagged cullins, as set forth above. Transfectedcells were metabolically labeled with [³⁵S]-methionine, and cell lysateswere immunoprecipitated reciprocally with either anti-HA, anti-CUL1 oranti-myc antibody (FIG. 3A). Neither the myc antibody cross-reacted withROC1 (e.g., lanes 2 and 3, FIG. 3A) nor the HA antibody cross-reactedwith the cullins (lane 12, FIG. 3A, and also lanes 6–9 of FIG. 4D). Allfive cullins were co-precipitated with ROC1 by the HA antibody. In thereciprocal immunoprecipitations, HA-ROC1 protein was detected readily inanti-myc-mCUL4A by the myc antibody, but was not evident inanti-myc-cullin 2, 3 and 5 immunocomplexes. Un-tagged CUL1 formed acomplex with co-transfected ROC1 with similar efficiency as myc taggedcullins (lane 1, FIG. 3A), excluding the possibility of any artifactualbinding between ROC1 and cullin proteins that might be caused by mycepitope tagging or cross-reactivity of the myc antibody. In addition tothe ROC1-cullin association, several cellular proteins of unknownidentity were precipitated with either ROC1 or a cullin protein,including an 130 kDa cellular protein (p130) that was co-precipitatedwith HA-ROC 1 when CUL1, but not other cullins, was co-expressed (FIG.3A, lane 7). The ROC1-cullins association in transfected cells has beenconfirmed by sequential immunoprecipitation and immunoblotting(IP-Western). Cullins 1, 2, 4A and 5 were readily detected in theanti-HA immunocomplex (data not shown)

To obtain evidence for in vivo ROC1-cullin association under morephysiological conditions, rabbit polyclonal antibodies specific to ROC1were raised. This antibody is capable of precipitating both ROC1 and theROC1-CUL1 complex as determined by the use of in vitro translatedproteins (lanes 1 and 2, FIG. 3B). From metabolically labeled HeLa andSaos-2 cells, the anti-ROC1 antibody precipitated a protein ofapproximately 14 kDa (lanes 3 and 5). This 14 kDa protein corresponds toROC1 as judged by its co-migration with in vitro produced ROC1 and bycompetition using the antigen peptide (lanes 4 and 6). In addition toROC1, a number of cellular proteins between 75 to 200 kDa wereco-precipitated with ROC1. The presence of these proteins in theanti-ROC1 immunocomplex is blocked by the competing antigen peptide,suggesting that these proteins may specifically associate with ROC1.

This observation suggests that in vivo ROC1 may be associated with manydifferent proteins, a conclusion consistent with its broad interactionwith all cullin proteins. To directly confirm this, several proteins atmolecular weights between 70 to 120 kDa were immunopurified from theROC1 immunocomplexes (indicated as “cullins” in the FIG. 3B) and theirsequences determined by protein microsequencing. At least four cullinproteins were identified from this analysis thus far; cullin 1 or cullin2 (KDVFQK, [SEQ ID NO: 25] corresponding to residues 459–464 in humanCUL1, database accession AF062536, or 428–433 of human CUL2, accessionQ13617), cullin 2 (KIFLENHVRHLH, [SEQ ID NO: 26] residues 62–73,accession Q13617), cullin 3 (KDVFERYY, residues 425–432 [SEQ ID NO: 27]and KVYTYVA, [SEQ ID NO: 28] residues 762–768, accession AF062537), andcullin 4A or 4B (KRIESLIDRDY, [SEQ ID NO: 29] residues 396–406 in humanCUL4A, accession Q13619 or residues 263–273 in human CUL4B, accessionQ13620). The association between ROC1 with many of these cullins was notdisrupted by the wash of immunocomplexes with a buffer containing 0.1%SDS (data not shown), indicating that the ROC1-cullins association isquite stable.

To further demonstrate the in vivo ROC1-cullins association withoutoverexpression, HeLa cell lysate was immunoprecipitated with antibodiesto ROC1, CUL1, and CUL2, and the precipitates analyzed by Westernblotting. As shown in FIG. 3C, both CUL1 (lane 3) and CUL2 (lane 7) werereadily detected in the ROC1 immunocomplexes and were specificallyblocked by the competing ROC1 antigen peptide. Reciprocally, ROC1 wasdetected in both CUL1 (lane 1) and CUL2 (lane 5) complexes (lower panel,FIG. 3C). Demonstration of association between ROC1 and other cullins byIP-Western was not carried out because of the lack of antibodies toother cullins at present.

EXAMPLE 10 Selective Interaction Between ROC2, APC11 and Cullin FamilyProteins

The yeast two-hybrid assay and in vivo binding assay described hereinwere used to determine whether ROC2 and APC11, like ROC1, also interactwith cullins. Full length human ROC2 or APC11 was fused in-frame withthe yeast Gal4 DNA activation domain and co-transformed into yeast cellswith individual cullins fused to the Gal4 DNA binding domain. Almostidentical to ROC1, ROC2 interacted strongly with cullins 1, 2, 4A and 5(FIG. 4A), indicating that ROC2 is also a general cullin-interactingprotein. In contrast, APC11 only interacted with cullin 5, but not othercullins (FIG. 4B).

To further asses the interaction between ROC2 and APC11 with cullins,Saos-2 cells were transfected with plasmids directing the expression ofHA tagged human ROC2 (HA-ROC2) or APC11 (HA-APC11) together withuntagged CUL1 or individual myc tagged cullins. Transfected cells weremetabolically labeled with [³⁵S]-methionine, and cell lysates wereimmunoprecipitated with either anti-HA, anti-CUL1 or anti-myc antibody(FIGS. 4C and 4D). Transfected HA-ROC2 protein migrates as a doublet(lanes 6 to 10, FIG. 4C). The myc antibody does not cross-react witheither form of ROC2 (e.g. comparing lanes 5 and 6). All five cullinswere co-precipitated with ROC2 by the HA antibody (lanes 6 to 10).Reciprocally, ROC2 (preferentially the faster migrating form) was alsodetected in cullin 2, 3 and 4 immunocomplexes (lanes 2 to 4).

In contrast and with the exception of cullin 5, APC11 and cullins werenot detected to interact with each other in reciprocal precipitations(lane 1 to 10, FIG. 4D). Cullin 5 was weakly, but reproducibly, detectedin the APC11 immunocomplex (lane 10). Of all six mammalian cullins, CUL5is the most divergent member of the cullin family and contains thehighest sequence similarity to APC2. In addition to the cullins, severalcellular proteins including a band of approximately 130 kDa was detectedin the ROC2 complex when CUL5, but not other cullins, was co-expressed(FIG. 4C, lanes 5 and 10). p130 was not detected in cells co-transfectedwith CUL5 and ROC1 (lane 11 of FIG. 3A) or APC11 (lane 10 of FIG. 4D).Whether this ROC2-CUL5-associated p130 is related to theROC1-CUL1-associated p130 (FIG. 3A, lane 7) and the functional rolesthese proteins may play in cullin-ROC complexes have not beendetermined. Cullin 2, 3 and 4 immunocomplexes, when precipitated fromcells co-transfected with ROC2, but not APC11, contained a cellularprotein of approximately 17 kda. The presence of this 17 kda polypeptidewas not evident in either CUL1 or CUL5 immunocomplexes which containedlittle ROC2, suggesting that its association with cullin 2–4 iscon-elated with, and may actually be dependent on or is promoted by, theassociation of cullins with ROC2.

EXAMPLE 11 ROC1 and ROC2 do not Interact with APC2

APC11 was co-purified with another APC subunit, APC2, which containslimited sequence similarity to cullins (Zachariae et al., 1998; Yu etal., 1998a, supra). When tested by the two-hybrid assay, APC11, but notROC1 nor ROC2, interacted with mouse APC2 in yeast cells (FIG. 4E). Toassess the interaction between APC2 and these three closely related RINGfinger proteins in mammalian cells, HeLa cells were transfected withplasmids directing the expression of myc-epitope tagged APC2 with eitherHA-epitope tagged ROC1, ROC2 or APC11, and determined their respectivebindings in vivo. Consistent with the yeast two-hybrid assay, APC2 andAPC11 were reciprocally detected in APC11 and APC2 immunocomplexes,respectively (data not shown). Weak binding was detected betweenectopically expressed ROC1 and APC2, but ROC2, even when overproduced,was not seen to interact with APC2 (data not shown).

EXAMPLE 12 Decrease of ROC1p Protein Causes a cdc53-, cdc34- andcdc4-Like Phenotype

The yeast genome contains a single ROC gene, Sc-ROC1 (ORF YOL133w), thatshares 67% sequence identity with human ROC1 (FIG. 2C), providing asimpler and more genetically facile system to determine the in vivofunction of ROC family proteins. The consequence of deleting the ScROC1gene by replacing it with a kanamycin resistance module was determinedby PCR homologous recombination. One copy of ScROC1 was replaced in adiploid, and the heterozygous yeast was subjected to sporulation andtetrad dissection (FIG. 5A). A 2:2 segregation was observed in 19 of 20tetrads dissected on complete medium, and all of the viable colonieswere kanamycin sensitive when replica plated onto selective medium (datanot shown). Upon microscopic inspection of the inviable spores,germination and a limited number of cell divisions to form microcolonieswere observed reflecting a “maternal” supply of ROC1p. Hence, ScROC1appears to be an essential gene for yeast viability.

A conditional yeast strain in which ScROC1 was under the control of thegalactose-inducible, glucose-repressible GAL1 promoter was created. AnHA3 tag was fused in-frame with the ScROC1 gene to monitor the level ofROC1 protein expression. Transformants were sporulated and dissected(2:2 segregation was observed), and haploid yeast containingGAL-HA3-ScROC1 were isolated and verified by PCR analysis (data notshown) and protein expression (FIG. 5C). High levels of expression ofHA3-ROC1p (FIG. 5B) or untagged ROC1p (data not shown) from the GAL1promoter had no detectable effect on yeast growth. Repression of ScROC1expression after switching to glucose resulted in a rapid decrease ofROC1p protein (FIG. 5C), suggesting that overexpressed ROC1p is anunstable protein with a short half life (˜t_(1/2)<20 minutes). Prolongedculturing of yeast cells in the presence of glucose, however, did notcompletely remove all of the ROC1 protein. A barely detectable amount ofROC1p was expressed for up to 24 hours when cultured in the presence ofglucose, indicating that ROC1p may be continually expressed at a lowlevel probably as the result of leakiness of the GALL promoter (FIG.5C). Decrease of ScROC1 expression caused the yeast to begin exhibitinga mutant phenotype at nine hours and resulted in the accumulation of amultiply elongated budded yeast population containing a single nucleusby 24 hours (FIG. 5B).

The ROC1p depletion-induced phenotype is indistinguishable from thosecaused by temperature sensitive mutations in the CDC53, CDC4 and CDC34genes (Mathias et al., 1996, supra). This result suggests that theScROC1 gene is involved in the same pathway as these genes incontrolling the ubiquitin-mediated proteolysis of proteins during the GIphase of the cell cycle such as CDK inhibitor p40Sic1p. To provideevidence supporting this conclusion, it was determined whether the yeastROC/APC11 family, like their human homologues, could directly interactwith the yeast cullin/CDC53 family by the yeast-two-hybrid system. Theyeast genome contains four cullin members, CDC53, CUL-B (ORF YGR003w),CUL-C(ORF YJL047c) and APC2. Each gene was fused in-frame with the Gal4DNA binding domain and co-transformed with ScROC1 or ScAPC11 fusedin-frame with the GAL4 activation domain. ScAPC2 was self-activating asa bait and was fused in-framed with the GAL4 activation domain andtested with ScROC1 fused in-frame with the DNA binding domain. ScROC1interacted with all four yeast cullin genes including the most distantlyrelated APC2 as determined by the activation of histidine reporter gene.In contrast, ScAPC11 only interacted weakly with CUL-C, but not CDC53 orCUL-B (FIG. 5D). Interaction of ScAPC11 with ScAPC2 could not be testedbecause both are self-activating as baits. Hence, like human ROCproteins, yeast ROC1 also commonly interacts with all members of cullinfamily proteins.

EXAMPLE 13 Functional Rescue of ROC1p Deficiency

Taking advantage of conditional phenotype induced by the depletion ofROC1p, the functional conservation and specificity of the ROC familyproteins was determined. The multi-budded phenotype incurred by ROC1pdepletion can be completely rescued by the expression of yeast ROC1, butnot vector control (FIG. 5E), confirming that the level of ROC1p was therate limiting factor causing the multi-budded phenotype. Ectopicexpression of both human ROC1 and ROC2 also rescued the phenotype ofScROC1p depletion, but less efficiently than yeast ROC1 as evidenced bya small fraction of cells still exhibiting the phenotype. This indicatesan evolutionary conservation of the ROC gene family and provides in vivoevidence supporting a function of human ROC1 in ubiquitin-mediatedproteolysis. Ectopic expression of yeast APC11, on the other hand, didnot rescue the phenotype caused by the decreased level of ROC1p (FIG.5E), demonstrating a functional specificity between members of theROC/APC11 family.

EXAMPLE 14 ROC1p is Required for SIC1p Degradation

A determination as to whether ScROC1 plays a role in regulating proteindegradation was based on the phenotypic similarity between ROC1pdepleted and cdc53 mutant cells and the interaction of ScROC1 withCDC53. A critical substrate of the CDC53 pathway is the G1 CDKinhibitor, p40Sic1p, which is targeted for ubiquitin mediateddegradation by the yeast SCF (Skowyra et al., 1997; Feldman et al.,1997, supra). To determine whether Sic1p was stabilized in yeastdepleted of ScROC1p, a yeast strain was created by PCR homologousrecombination in which the SIC1 gene in the GAL-HA3-ScROC1 yeast wasepitope tagged with HA3. Yeast cells grown in a low concentration ofgalactose (0.05% plus 2% raffinose), expressing a reduced level of ROC1pbut still exhibiting a wild type phenotype, were switched to glucosemedia for different lengths of time to deplete the expression of ROC1p.Appearance of the multiple budded phenotype was confirmed by microscopicexamination. Total cells lysates were prepared from samples collectedfrom each time point and subjected to western analysis. The ROC1pprotein was depleted and became almost undetectable after culturing inglucose media at the 9 hour point (data not shown). Closely correlatedwith the appearance of multiple elongated buds, Sic1 protein accumulatedafter 14 hours of culturing in glucose and was sustained at a high levelthroughout the experimental period (FIG. 5F). An anti-actin antibody wasused to confirm the equal loading of proteins from different time points(FIG. 5F). These results provide in vivo evidence that ROC1 functions inubiquitin-mediated proteolysis.

EXAMPLE 15 ROC1 is a Critical Subunit of Cullin Ubiquitin LigaseActivity

CDC53, the closest yeast homologue of human CUL1, assembles into afunctional E3 ubiquitin ligase complex in insect cells with E2 CDC34,SKP1 and an F box protein (SCF complex) to catalyze ubiquitination ofphosphorylated substrates (Skowyra et al., 1997; Feldman et al., 1997,supra). Protein complexes containing human CUL1, SIP1 and SKP2 assembledin insect cells, however, were found to contain little ubiquitin ligaseactivity, but became active after incubating with HeLa cell lysate(Lyapina et al., 1998, supra), raising the possibility that anadditional rate limiting component(s) is required for cullin-dependentubiquitin ligase activity. To determine whether ROC1 may functionbiochemically as a subunit of ubiquitin ligase activity, the ubiquitinligation activity of the ROC1 and CUL1 immunocomplexes was analyzed.293T cells were transiently transfected with plasmid DNA expressing HAepitope tagged ROC1 (HA-ROC1) and cullin 1 and ROC1-CUL1 complex wasrecovered by immunoprecipitation using anti-HA antibody. To facilitatethe recovery of functional ROC1- and cullin 1-associated ubiquitinligase complex, the F-box protein SKP2, which has been previouslydemonstrated to interact with CUL1, was included in the transfection.SKP1, which mediates the binding of CUL1 with SKP2, is expressed at highlevel in the cell and was not included in the transfection. Theubiquitin ligase activity of ROC1 and CUL1 was measured by incubatingthe HA-ROC1-CUL1 immunocomplex immobilized on protein A agarose beadswith purified human E1, mouse E2 CDC34, ATP and ³²P-labeled ubiquitin(Ub). After incubation, the reactions were terminated by boiling thesamples in the presence of SDS and reducing agent and mixtures wereresolved by SDS-PAGE, followed by autoradiography. An evident,time-course dependent ubiquitin ligation, as visualized by theincorporation of ³²P-Ub into covalently linked high molecular weightsmear characteristic of ubiquitinated proteins, was detected when bothE1 and E2 CDC34 were added to the HA-ROC1/CUL1/SKP2 immunocomplexes(lane 1, lanes 4 to 9, FIG. 6A), but not when either E1 (lane 2) or E2(lane 3) was omitted, indicating an E1 and E2 dependent-ubiquitinligation. As a control, anti-HA precipitate derived from cellstransfected without a HA-tagged protein exhibited only E1- or E2-linkedmono-ubiquitin conjugates (lanes 5 and 8, FIG. 6B). The observed proteinladder reflects an increment of a single 32P-Ub (˜12 kDa in the form ofa recombinant protein), a characteristic of ubiquitination reaction. Thetreatment of the reaction mixture with DTT, SDS and boilingsignificantly reduced, but cannot completely abolish the Ub-E1 (markedas 32P-Ub-E1, FIG. 6) and Ub-CDC34 (marked as 32P-Ub-CDC34) conjugates.No exogenous substrate protein was added to the reaction. Accumulationof high molecular weight ubiquitinated proteins could therefore beresulted from either the ubiquitination of a SKP2-targeted substrate(s)co-precipitated with the HA-ROC1 complex or a ligation of ubiquitinproteins. A careful examination of molecular weight increment from theubiquitination reaction indicates that the ROC1-CUL1 complex cancatalyze ubiquitin ligation independent of a substrate, and most, if notall high molecular weight masses correspond to polyubiquitin chainsconsisting of a series of ubiquitin molecules without an attachedsubstrate (data not shown).

To determine the contribution of individual proteins to the ubiquitinligase activity in the HA-ROC1 immunocomplex, a series of “drop-out”transfections was performed. Omission of SKP2, an F-box protein thatpresumably brings substrate protein(s) to CUL1, only slightly reducedthe ubiquitin ligase activity (comparing lanes 2 and 3, FIG. 6B). Such anon-essential role of transfected SKP2 to the ubiquitin ligase activityof the HA-ROC1 complex may be due in part to the presence of endogenousSKP2 in 293 cells (Zhang, H., et al., (1995) Cell 82, 915–925), orindicating a substrate-independent ligation of ubiquitin molecules.Omission of CUL1, however, severely reduced the ubiquitin ligaseactivity of the ROC1 immunocomplex (lane 4). Reciprocally, omission ofROC1 from the CUL1 complex, like the omission of CUL1 from ROC1 complex,also significantly reduced ubiquitin ligase activity (comparing lanes 6and 7). There was a low level of ligase activity in the CUL1immunocomplex without co-transfection with ROC1, likely resulted fromthe endogenous ROC1 protein. These results indicate an inter-dependencyof ROC1- and CUL1-associated ubiquitin ligase activity upon theexpression of both proteins, suggesting that ROC1 and CUL1 act asintegral parts of an E3 ubiquitin ligase.

EXAMPLE 16 In Vivo Ubiquitin Ligase Activity of ROC1 and CUL1

To directly demonstrate a ROC1 associated ubiquitin ligase activity invivo, ROC1 and CUL1 complexes from either 293T or HeLa cells wereimmunoprecipitated using affinity purified antibody specific to eitherprotein and assayed for their ability to catalyze ubiquitin ligation(FIG. 6C). Like the HA-ROC1 immunocomplex precipitated from transfectedcells, the ROC1 immunocomplex derived from both HeLa (lane 3) and 293Tcells (lane 7) actively catalyzed the incorporation of ³²P-labeledubiquitin into high molecular weights in an E1 (lane 1) and E2 CDC34(lane 2) dependent manner. Similarly, the CUL1 complex also exhibited ahigh level of ubiquitin ligase activity (lane 6). In contrast, theanti-APC11 complex exhibited only background levels of ligase activitywhen similarly incubated with E1 and E2 CDC34 (lane 5). It has this beendetermined that the anti-APC11 antibody is capable of precipitatingAPC11 as well as a number of additional cellular proteins, likelycorresponding to other components of the APC complex (data not shown).These results, together with in vitro biochemical analysis demonstratingthe catalytic role of the CUL1-ROC1 dimeric complex (Tan et al.,accompanying paper), indicate that ROC1 is an essential subunit ofcullin associated ubiquitin ligase.

EXAMPLE 17 Summation of Experimental Results

Four lines of evidence provided herein demonstrate that the ROC familyproteins function as essential subunits of cullin ubiquitin ligases.First, both ROC1 and ROC2 interact directly with all five mammaliancullins that we have examined as determined by several different assaysboth in vitro and in vivo (FIGS. 1 and 3). Conventional biochemicalpurification has further identified ROC1 as a stoichiometricallyassociated subunit of CUL1 ubiquitin ligase activity (Tan et al.,accompanying paper). Further underscoring the generality of this binaryinteraction is the parallel association in the APC E3 ligase between acullin-related protein, APC2, and a ROC homologous protein, APC11 (FIG.4). Among more than a dozen subunits identified, ROC/APC11 andcullin/APC2 are the only two proteins common between the APC and the SCFcomplexes.

Second, the examples set forth above demonstrate that ROC1 is essentialfor cullin function in vivo. Yeast ROC1 is an essential gene whosedepletion results in a multiple elongated bud phenotypeindistinguishable from that caused by cdc53, cdc34 and cdc4 mutationsand results in accumulation of the CDK inhibitor Sic1 as in cdc53, cdc34and cdc4 mutants. Similarly, ROC-related APC11 has been shown to be anessential subunit for APC function. Loss of APC11 function in yeastresulted in accumulation of APC substrates and caused metaphase arrest(Zachariae et al., 1998, supra).

Third, the examples above illustrate that ROC1 is an essential subunitof cullin ubiquitin ligase. ROC1 and cullin 1 immunocomplexesprecipitated from in vivo catalyze ligation of ubiquitins to formpolyubiquitin chains. Omission of ROC1 dramatically reduced ubiquitinligase activity from the CUL1 immunocomplex (FIG. 6).

Finally, an in vitro ROC1 and CUL1 ubiquitin ligase activity that isspecifically dependent on E1 and E2 has been reconstructed (Tan et al.,accompanying paper).

One ramification of the findings set forth herein is that APC11 (ROC1homologue) and APC2 (homologous to cullins) is the ligase in the APC.The extensive studies on ubiquitin-mediated proteolysis during themitotic phase of the cell cycle have identified the APC as the singlemajor E3 ubiquitin ligase required to degrade most mitotic regulatoryproteins. Recently, yeast CDC53 has been identified as a major E3 ligaseactivity regulating S phase entry. Though the in vivo function of mostcullins are yet to be determined, some may well perform other functionsunrelated to cell cycle control. Although the ubiquitin ligase core ofboth APC and SCF complexes share structural similarities, one containsAPC11 and APC2, the other involves CUL1 and ROC1, the two ligasesexhibit an evident specificity. While both ROC proteins commonlyinteract with all cullins, APC11 specifically interacts with APC2.Functional support to this specificity comes from the finding that whileboth human ROC1 and ROC2 are capable of functionally rescuing thephenotype caused by the depletion of yeast ROC1, yeast APC11 can not(FIG. 5). Hence, ROC-cullin and APC11-APC2 function separately duringinterphase and mitosis, respectively. Furthermore, there exist twodistinct ROC proteins in higher eukaryotes, both capable of directlyinteracting with all members of the cullin family. Their combinatorialinteractions with different cullins point to a potentially large numberof ubiquitin ligases, and each may be involved in a specific cellularpathway as in the case of the SCF and APC complexes, perhaps reflectingthe complexity of interphase regulation.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. An isolated polynucleotide comprising a nucleic acid encodingRegulator of Cullins 1 (ROC1), said nucleic acid selected from the groupconsisting of: (a) a nucleic acid consisting of the nucleotide sequenceof SEQ ID NO:1; (b) a nucleic acid which encodes a protein that forms acomplex with a cullin protein and/or has ubiquitin ligase activity,wherein said nucleic acid hybridizes to the complete complement of anucleic acid consisting of the nucleotide sequence of SEQ ID NO:1 understringent conditions defined by a wash of 50% Formamide, 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C.; (c) a nucleic acid whichencodes a protein that forms a complex with a cullin protein and/or hasubiquitin ligase activity, wherein said nucleic acid has at least 95%sequence identity to the nucleotide sequence of SEQ ID NO:1; and (d) anucleic acid that differs from the nucleic acid of (a) to (c) above dueto the degeneracy of the genetic code.
 2. An isolated polynucleotideaccording to claim 1, wherein said nucleic acid encodes a ROC1 proteinconsisting of the amino acid sequence of SEQ ID NO:2.
 3. An isolatedpolynucleotide according to claim 1, wherein said nucleic acid consistsof the nucleotide sequence of SEQ ID NO:1.
 4. An expression vectorcomprising the isolated polynucleotide of claim
 1. 5. A cell comprisingthe expression vector of claim
 4. 6. The cell of claim 5, wherein saidcell is capable of expressing said nucleic acid encoding ROC1.
 7. Anantisense oligonucleotide that is 12 to 50 nucleotides in length and iscompletely complementary to a portion of the nucleic acid of claim
 1. 8.The antisense oligonucleotide of claim 7, wherein said oligonucleotideis DNA.
 9. An expression vector capable of transcribing the antisenseoligonucleotide of claim
 7. 10. A method for producing a proteincomprising the amino acid sequence of SEQ ID NO:2, comprising (a)culturing a host cell comprising an expression vector comprising apolynucleotide comprising a nucleic acid selected from the groupconsisting of: (i) a nucleic acid consisting of the nucleotide sequenceof SEQ ID NO:1; and (ii) a nucleic acid that differs from the nucleicacid of (i) above due to the degeneracy of the genetic code; and (b)recovering the protein from the host cell culture.
 11. A method forproducing a peptide or protein, comprising: (a) culturing a host cellcomprising an expression vector comprising a polynucleotide consistingof a segment of at least 60 consecutive nucleotides of a nucleic acidselected from the group consisting of: (i) a nucleic acid consisting ofthe nucleotide sequence of SEQ ID NO:1; and (ii) a nucleic acid thatdiffers from the nucleic acid of (i) above due to the degeneracy of thegenetic code; and (b) recovering the peptide from the host cell culture.